Amyloid binding nitrogen-linked compounds for the antemortem diagnosis of alzheimer&#39;s disease, in vivo imaging and prevention of amyloid deposits

ABSTRACT

Amyloid binding compounds which are derivatives of Chrysamine G, pharmaceutical compositions containing, and methods using such compounds to identify Alzheimer&#39;s brain in vivo and to diagnose other pathological conditions characterized by amyloidosis, such as Down&#39;s Syndrome are described. Pharmaceutical compositions containing Chrysamine G and derivatives thereof and methods using such compositions to prevent cell degeneration and amyloid-induced toxicity in amyloidosis associated conditions are also described. Methods using Chrysamine G derivatives to stain or detect amyloid deposits in biopsy or post-mortem tissue are also described. Methods using Chrysamine G derivatives to quantify amyloid deposits in homogenates of biopsy and post-mortem tissue are also described.

This application is a continuation in part of U.S. application Ser. No.08/640,704, filed May 1, 1996, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 08/432,019,filed May 1, 1995, now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 08/282,289, filed Jul. 29, 1994, nowabandoned.

The present invention was made utilizing funds from the NationalInstitute of Ageing, grant numbers AG-05443, and AG-05133 and AG-08974.

BACKGROUND OF THE INVENTION

The present invention relates to the identification of compounds thatare suitable for imaging amyloid deposits in living patients. Morespecifically, the present invention relates to a method of imagingamyloid deposits in brain in vivo to allow antemortem diagnosis ofAlzheimer's Disease. The present invention also relates to therapeuticuses for such compounds.

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age, yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

By definition, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortemslices of brain tissue of victims of Alzheimer's disease exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD.

The amyloid cores of these neuritic plaques are composed of a proteincalled the β-amyloid (Aβ) that is arranged in a predominatelybeta-pleated sheet configuration. Mori et al., Journal of BiologicalChemistry 267: 17082 (1992); Kirschner et al., PNAS 83: 503 (1986).Neuritic plaques are an early and invariant aspect of the disease. Mannet al., J. Neurol. Sci. 89: 169; Mann, Mech. Ageing Dev. 31: 213 (1985);Terry et al., J. Neuropathol. Exp. Neurol 46: 262 (1987).

The initial deposition of Aβ probably occurs long before clinicalsymptoms are noticeable. The currently recommended “minimum microscopiccriteria” for the diagnosis of AD is based on the number of neuriticplaques found in brain. Khachaturian, Arch. Neurol., supra (1985).Unfortunately, assessment of neuritic plaque counts must be delayeduntil after death.

Amyloid-containing neuritic plaques are a prominent feature of selectiveareas of the brain in AD as well as Downs Syndrome and in personshomozygous for the apolipoprotein E4 allele who are very likely todevelop AD. Corder et al., Science 261: 921 (1993); Divry, P., J.Neurol. Psych. 27: 643-657 (1927); Wisniewski et al., in Zimmerman, H.M. (ed.): PROGRESS IN NEUROPATHOLOGY, (Grune and Stratton, N.Y. 1973)pp. 1-26. Brain amyloid is readily demonstrated by staining brainsections with thioflavin S or Congo red. Puchtler et al., J. Histochem.Cytochem. 10: 35 (1962). Congo red stained amyloid is characterized by adichroic appearance, exhibiting a yellow-green polarization color. Thedichroic binding is the result of the beta-pleated sheet structure ofthe amyloid proteins. Glenner, G. N. Eng. J. Med. 302: 1283 (1980). Adetailed discussion of the biochemistry and histochemistry of amyloidcan be found in Glenner, N. Eng. J. Med., 302: 1333 (1980).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include (1) genetic testing, (2) immunoassay methods and (3)imaging techniques.

Evidence that abnormalities in β metabolism are necessary and sufficientfor the development of AD is based on the discovery of point mutationsin the Aβ precursor protein in several rare families with an autosomaldominant form of AD. Hardy, Nature Genetics 1: 233 (1992); Hardy et al.,Science 256: 184 (1992). These mutations occur near the N- andC-terminal cleavage points necessary for the generation of Aβ from itsprecursor protein. St. George-Hyslop et al., Science 235: 885 (1987);Kang et al., Nature 325: 733 (1987); Potter WO 92/17152. Geneticanalysis of a large number of AD families has demonstrated, however,that AD is genetically heterogeneous. St. George-Hyslop et al., Nature347: 194 (1990). Linkage to chromosome 21 markers is shown in only somefamilies with early-onset AD and in no families with late-onset AD. Morerecently a gene on chromosome 14 whose product is predicted to containmultiple transmembrane domains and resembles an integral membraneprotein has been identified by Sherrington et al., Nature 375: 754-760(1995). This gene may account for up to 70% of early-onset autosomaldominant AD. Preliminary data suggests that this chromosome 14 mutationcauses an increase in the production of Aβ. Scheuner et al., Soc.Neurosci. Abstr. 21: 1500 (1995). A mutation on a very similar gene hasbeen identified on chromosome 1 in Volga German kindreds withearly-onset AD. Levy-Lahad et al., Science 269: 973-977 (1995).

Screening for apolipoprotein E genotype has been suggested as an aid inthe diagnosis of AD. Scott, Nature 366: 502 (1993); Roses, Ann. Neurol.38: 6-14 (1995). Difficulties arise with this technology, however,because the apolipoprotein E4 allele is only a risk factor for AD, not adisease marker. It is absent in many AD patients and present in manynon-demented elderly people. Bird, Ann. Neurol. 38: 2-4 (1995).

Immunoassay methods have been developed for detecting the presence ofneurochemical markers in AD patients and to detect an AD related amyloidprotein in cerebral spinal fluid. Warner, Anal. Chem. 59: 1203A (1987);World Patent No. 92/17152 by Potter; Glenner et al., U.S. Pat. No.4,666,829. These methods for diagnosing AD have not been proven todetect AD in all patients, particularly at early stages of the diseaseand are relatively invasive, requiring a spinal tap. Also, attempts havebeen made to develop monoclonal antibodies as probes for imaging of Aβ.Majocha et al., J. Nucl. Med., 33: 2184 (1992); Majocha et al., WO89/06242 and Majocha et al., U.S. Pat. No. 5,231,000. The majordisadvantage of antibody probes is the difficulty in getting these largemolecules across the blood-brain barrier. Using antibodies for in vivodiagnosis of AD would require marked abnormalities in the blood-brainbarrier in order to gain access into the brain. There is no convincingfunctional evidence that abnormalities in the blood-brain barrierreliably exist in AD. Kalaria, Cerebrovascular & Brain MetabolismReviews 4: 226 (1992).

Aβ antibodies are also disadvantageous for use in AD diagnostics becausethey typically stain diffuse deposits of Aβ in addition to the neuriticplaques—and the diffuse plaques often predominate. Yamaguchi et al.,Acta Neuropathol., 77: 314 (1989). Diffuse plaques may be a separatetype of lesion, not necessarily involved in the dementing process of AD.The latter is suggested by findings of diffuse plaques in cognitivelynormal controls and aged dogs. Moran et al., Medicina Clinica 98: 19(1992); Shimada et al., Journal of Veterinary Medical Science 54: 137(1992); Ishihara et al., Brain Res. 548: 196 (1991); Giaccone et al.,Neurosci. Lett. 114: 178 (1990). Even if diffuse plaques are forerunnersof neuritic plaques, the key pathological event in AD may be the processthat turns the apparently benign diffuse plaque into the neuritic plaquewith its associated halo of degeneration. Therefore, a probe is neededthat is specific for the neuritic plaque and NFTs as a more specificmarker for AD pathophysiology than antibodies that would also labeldiffuse plaques.

Recently, radiolabeled Aβ peptide has been used to label diffuse,compact and neuritic type plaques in sections of AD brain. Maggio etal., WO 93/04194. However, these peptides share all of the disadvantagesof antibodies. Specifically, peptides do not normally cross theblood-brain barrier in amounts necessary for imaging and because theseprobes react with diffuse plaques, they may not be specific for AD.

Congo red may be used for diagnosing amyloidosis in vivo in non-brainparenchymal tissues. However, Congo red is probably not suitable for invivo diagnosis of Aβ deposits in brain because only 0.03% of an injecteddose of iodinated Congo red can enter the brain parenchyma. Tubis etal., J. Amer. Pharm. Assn. 49: 422 (1960). Radioiodinatedbisdiazobenzidine compounds related to Congo red, such as Benzo Orange Rand Direct Blue 4, have been proposed to be useful in vitro and in vivoto detect the presence and location of amyloid deposits in an organ of apatient. Quay et al., U.S. Pat. Nos. 5,039,511 and 4,933,156. However,like Congo red, all of the compounds proposed by Quay contain stronglyacidic sulfonic acid groups which severely limit entry of thesecompounds into the brain making it extremely difficult to attain animaging effective quantity or detectable quantity in the brainparenchyma.

The inability to assess amyloid deposition in AD until after deathimpedes the study of this devastating illness. A method of quantifyingamyloid deposition before death is needed both as a diagnostic tool inmild or clinically confusing cases as well as in monitoring theeffectiveness of therapies targeted at preventing Aβ deposition.Therefore, it remains of utmost importance to develop a safe andspecific method for diagnosing AD before death by imaging amyloid inbrain parenchyma in vivo. Even though various attempts have been made todiagnose AD in vivo, currently, there are no antemortem probes for brainamyloid. No method has utilized a high affinity probe for amyloid thathas low toxicity, can cross the blood-brain barrier, and binds moreeffectively to AD brain than to normal brain in order to identify ADamyloid deposits in brain before a patient's death. Thus, no in vivomethod for AD diagnosis has been demonstrated to meet these criteria.

Very recent data suggest that amyloid-binding compounds will havetherapeutic potential in AD and type 2 diabetes mellitus. As mentionedabove, there are two broad categories of plaques in AD brain, diffuseand neuritic (classical). Diffuse plaques do not appear to inducemorphological reactions such as the reactive astrocytes, dystrophicneurites, microglia cells, synapse loss, and full complement activationfound in neuritic plaques. Joachim et al., Am. J. Pathol. 135: 309(1989); Masliah et al., loc. cit. 137: 1293 (1990); Lue and Rogers,Dementia 3: 308 (1992). These morphological reactions all signify thatneurotoxic and cell degenerative processes are occurring in the areasadjacent to the compact Aβ deposits of neuritic plaques. Aβ-inducedneurotoxicity and cell degeneration has been reported in a number ofcell types in vitro. Yankner et al., Science 250: 279 (1990); Roher etal., BBRC 174: 572 (1991); Frautschy et al., Proc. Natl. Acad. Sci. 88:83362 (1991); Shearman et al., loc. cit. 91: 1470 (1994). It has beenshown that aggregation of the Aβ peptide is necessary for in vitroneurotoxicity. Yankner, Neurobiol. Aging 13: 615 (1992). Differences inthe state of aggregation of Aβ in diffuse and neuritic plaques mayexplain the lack of neurotoxic response surrounding the diffuse plaque.Lorenzo and Yankner, Proc. Natl. Acad. Sci., 91: 12243 (1994). Recently,three laboratories have reported results which suggest that Congo redinhibits Aβ-induced neurotoxicity and cell degeneration in vitro.Burgevin et al., NeuroReport 5: 2429 (1994); Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994); Pollack et al., Neuroscience Letters184: 113 (1995); Pollack et al., Neuroscience Letters 197: 211 (1995).The mechanism appears to involve both inhibition of fibril formation andprevention of the neurotoxic properties of formed fibrils. Lorenzo andYankner, Proc. Natl. Acad. Sci. 91: 12243 (1994). Congo red also hasbeen shown to protect pancreatic islet cells from the toxicity caused byamylin. Lorenzo and Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994).Amylin is a fibrillar peptide similar to Aβ which accumulates in thepancreas in type 2 diabetes mellitus.

It is known in the art that certain azo dyes may be carcinogenic. Morganet al. Environmental Health Perspectives, 102(supp.) 2: 63-78, (1994).This potential carcinogenicity appears to be based largely on the factthat azo dyes are extensively metabolized to the free parent amine byintestinal bacteria. Cerniglia et al., Biochem. Biophys. Res. Com., 107:1224-1229, (1982). In the case of benzidine dyes (and many othersubstituted benzidines), it is the free amine which is the carcinogen.These facts have little implications for amyloid imaging studies inwhich an extremely minute amount of the high specific activityradiolabelled dye would be directly injected into the blood stream. Inthis case, the amount administered would be negligible and the dye wouldby-pass the intestinal bacteria.

In the case of therapeutic usage, these facts have critical importance.Release of a known carcinogen from a therapeutic compound isunacceptable. A second problem with diazo dye metabolism is that much ofthe administered drug is metabolized by intestinal bacteria prior toabsorption. This lowered bioavailability remains a disadvantage even ifthe metabolites released are innocuous.

Thus, a need exists for amyloid binding compounds which are similar toCongo red but which enter the brain (Congo Red does not). Such compoundscould be used in preventing cell degeneration associated with fibrilformation and thereby treat pathological conditions in amyloidassociated diseases, such as AD and Downs Syndrome and in treatingpancreatic islet cell toxicity in type 2 diabetes mellitus.

A further need exists for amyloid binding compounds that are non-toxicand bioavailable and, consequently, can be used in therapeutics.

SUMMARY OF THE INVENTION

It therefore is an object of the present invention to provide a safe,specific method for diagnosing AD before death by in vivo imaging ofamyloid in brain parenchyma. It is another object of the presentinvention to provide an approach for identifying AD amyloid deposits inbrain before a patient's death, using a high-affinity probe for amyloidwhich has low toxicity, can cross the blood-brain barrier, and candistinguish AD brain from normal brain. It is another object to providea treatment for AD which will prevent the deposition or toxicity of Aβ.It is another object to provide a technique for staining and detectingamyloid deposits in biopsy or post-mortem tissue specimens. It isanother object to provide a method for quantifying amyloid deposition inhomogenates of biopsy or post-mortem tissue specimens.

In accomplishing these and other objects, there has been provided, inaccordance with one aspect of the present invention, an amyloid bindingcompound of Formula I or a water soluble, non-toxic salt thereof:

wherein:

is either N═N—Q, CR′═N—Q, N═CR′—Q, CR′₂—NR′—Q, NR′—CR′₂—Q, (CO)—NR′—Q,NR′—(CO)—Q or NR′—NR′—Q (where R′ represents H or a lower alkyl group);

X is C(R″)₂

(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂O(CO)R′, OR′, SR′, COOR′, R_(Ph), CR′═CR′—R_(Ph),CR′₂—CR′₂—R_(Ph) (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R″), a tri-alkyl tin, atetrazole or oxadiazole of the form:

wherein R′ is H or a lower alkyl group)

or X is CR′═CR′, N═N, C═O, O, NR′ (where R′ represents H or a loweralkyl group), S, or SO₂;

each R₁ and R₂ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin, R_(Ph),CR′═CR′—R_(Ph), CR′₂—CR′₂—R_(Ph) (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R₁ andR₂), a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

 (wherein R₈ and R₉ are lower alkyl groups) or

and at least one of R₂ is not H, OCH₃, CH₃, or halogen when the compoundof Formula I is a 1,4-diazobenzene compound;

each Q is independently selected from one of the following structures,each of which contains a carboxylic acid or an acid-like functionality:

IA, IB, IC, ID, IE, IF and IG, wherein

IA has the following structure:

 wherein:

each of R₃, R₄, R₅, R₆ or R₇ independently is defined the same as R₁above and, wherein at least one of R₃, R₄, R₅, R₆, or R₇ is a hydroxy,sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's, andwherein at least one of R₁, or R₂ is a halogen when the compound ofFormula I is a 4,4′-diazobiphenyl compound;

IB has the following structure:

 wherein:

each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently is definedthe same as R₁ above, and wherein at least one of R₁₀, R₁₁, R₁₂, R₁₃,R₁₄, R₁₅ or R₁₆ is a hydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂ orcarboxy in both Q's, and wherein at least one of R₁, or R₂ is a halogenwhen the compound of Formula I is a 4,4′-diazobiphenyl compound;

IC has the following structure:

 wherein:

each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁ is defined the same as R₁ above;

ID has the following structure:

 wherein:

each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁ aboveand

represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein:

each of R₂₅, R₂₆, or R₂₇ independently is defined the same as R₁ aboveand

represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein:

exactly one of R₂₈, R₂₉, R₃₀, R₃₁ or R₃₂ is the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁ orR₃₂ independently is defined the same as R₁ above, and wherein at leastone of R₂₈, R₂₉, R₃₀, R₃₁ or R₃₂ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂ or carboxy in both Q's;

IG has the following structure:

 wherein:

exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈, or R₃₉ is the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈, or R₃₉ independently is defined the same as R₁ above, andwherein at least one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈, or R₃₉ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂ or carboxy in both Q's;

or wherein:

is NR′—N═Q (where R′ represents H or a lower alkyl group) and each

 is independently selected from one of the following structures:

IIA or IIB, wherein:

IIA has the following structure:

 wherein:

each of R₄₀-R₄₇ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂O(CO)R′, OR′, SR′, COOR′, R_(Ph), CR′═CR′—R_(Ph),CR′₂—CR′₂—R_(Ph) (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R₁), a tri-alkyl tin, atetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group).

IIB has the following structure:

 wherein:

each of R₄₈ and R₄₉ independently is a lower alkyl group, (CH₂)_(n)OR′where n=1, 2, or 3, CF₃, CH₂—CH₂F, CH₂—CH₂—CH₂F, CN, (C═O)—R′, NO₂,(C═O)N(R′)₂, COOR′, (C═O)—(CH₂)_(n)—(C₆H₅) where n=1, 2, or 3, R_(Ph),CR′═CR′—R_(Ph), CR′₂—CR′₂—R_(Ph) (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R₁), atetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group),

wherein at least one of R₄₈ or R₄₉ is (C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′,CN, or a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group) in both Q's.

The acid-like functionality of each Q is preferably provided byfunctional groups which contain an ionizable proton with a pK_(a) ofless than 10, preferably of from 2 to 9 and more preferably of from 4 to8. The functional groups may be selected from the group consisting ofhydroxy, sulfhydryl, tetrazole, oxadiazole, cylic amide,isoindole-1,3(2H)-dione, benzisoxazole,2,3-benzodiazine-1,4(2H,3H)-dione, 2,3-benzoxazine-1,4(3H)-dione,(2H)1,3-benzoxazine-2,4(3H)-dione, (3H)2-benzazine-1,3(2H)-dione, orNO₂.

The lower alkyl groups are preferably selected from the group consistingof methyl, ethyl, or straight-chain, branched, or cyclic propyl, butyl,pentyl or hexyl.

One embodiment of the present invention provides an amyloid bindingcompound of Formula I, as defined above, or a water soluble, non-toxicsalt thereof, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉is selected from the group consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F,¹⁹F, ¹²⁵I, CH₂—CH₂—¹⁸F, O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂—¹⁸F,O—CH₂—CH₂—CH₂—¹⁸F and a carbon-containing substituent as specified inFormula I wherein at least one carbon is ¹¹C or ¹³C.

The invention also provides an amyloid binding compound of Formula I, asdefined above, or a water-soluble, non-toxic salt thereof, wherein thecompound binds to Aβ with a dissociation constant (K_(D)) between 0.0001and 10.0 μM when measured by binding to synthetic Aβ peptide orAlzheimer's Disease brain tissue.

Another aspect of the present invention is to provide a method forsynthesizing an amyloid binding compound of Formula I, as defined above,or a water soluble, non-toxic salt thereof, wherein at least one of thesubstituents R₁-R₇ and R₁₀-R₄₉ is selected from the group consisting of¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F and ¹⁹F, comprising the step of reacting anamyloid binding compound of Formula I, as defined above, or a watersoluble, non-toxic salt thereof wherein at least one of the substituentsR₁-R₇ and R₁₀-R₄₉ is a tri-alkyl tin, with a halogenating agentcontaining ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F.

An additional aspect of the present invention is a pharmaceuticalcomposition for in vivo imaging of amyloid deposits, comprising (a) anamyloid binding compound of Formula I, as defined above, or a watersoluble, non-toxic salt thereof, wherein at least one of thesubstituents R₁-R₇ and R₁₀-R₄₉ is selected from the group consisting of¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F and a carbon-containing substituent asspecified in Formula I wherein at least one carbon is ¹¹C or ¹³C, and(b) a pharmaceutically acceptable carrier.

Yet another aspect of the present invention is an in vivo method fordetecting amyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of the above pharmaceuticalcomposition, and (b) detecting the binding of the compound to amyloiddeposit in said subject. A related aspect of the present inventionprovides an in vivo method for detecting amyloid deposits in a subjectwherein the amyloid deposit is located in the brain of a subject. Thismethod of the invention may be used in a subject who is suspected ofhaving an amyloidosis associated disease or syndrome selected from thegroup consisting of Alzheimer's Disease, Down's Syndrome, andhomozygotes for the apolipoprotein E4 allele.

Another aspect of the invention relates to pharmaceutical compositionsand methods of preventing cell degeneration and toxicity associated withfibril formation in amyloidosis associated conditions such as AD andType 2 diabetes mellitus. Such pharmaceutical compositions compriseChrysamine G or the above described derivatives thereof and apharmaceutically acceptable carrier. Such compounds would be non-toxic.

Another aspect of the invention relates to the use of the probes as astain for the visualization and detection of amyloid deposits in biopsyor post-mortem tissue specimens.

Another aspect of this invention relates to the use of radiolabeledprobes for the quantitation of amyloid deposits in biopsy or postmortemtissue specimens.

Another aspect relates to a method of distinguishing Alzheimer's diseasebrain from normal brain.

Other aspects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates the chemical structures of Chrysamine G and severalChrysamine G analogues or derivatives which have been synthesized andtested, including the 3-iodo derivative (3-ICG), the 3-iodo dimethoxyderivative (3-ICG(OMe)₂), the dimethyl ester derivative (CG(COOMe)₂),the phenol derivative, salicylic acid (SA), the aniline derivative(1/2CG), Congo red, the 3,3′-diiodo derivative (3,3′-I₂CG), the3,3′-dibromo derivative (3,3′-Br₂CG), the 3,3′-dichloro derivative(3,3′-Cl₂CG), the 3-bromo derivative (3-BrCG), and the 5-fluorosalicylicacid derivative ((5-FSA)CG). The numbers in the figure refer to eachcompound's K_(i) (in μM) for inhibition of [¹⁴C]Chrysamine G binding tothe synthetic peptide, Aβ (10-43).

FIG. 1B illustrates the chemical structures of several Chrysamine Ganalogues or derivatives which have been synthesized and tested,including the 3,3′-dicarboxylic acid derivative (3,3′-(COOH)₂CG), the2,2′-disulfonic acid derivative of Chrysamine G (2,2′-(SO₃)₂CG), the3-bromo, 3-isopropylsalicylic acid derivative (3-Br-(3-iPrSA)CG), the3-isopropylsalicylic acid derivative ((3-iPrSA)CG), the 2,4-diphenolderivative (2,4-Diphenol), the γ-resorcylic acid derivative((6-OHSA)CG), the 3,3′,5,5′-tetramethylbenzidine derivative(3,3′,5,5′-(CH₃)₄CG), the 3,3′-dimethyl derivative (3,3′-(CH₃)₂CG), the2,2′-dimethyl derivative(2,2′-(CH₃)₂CG), the benzisoxazole derivative(CG Benzisoxazole), and the 3-carboxy alkyne derivative (3-(COOH)—C3C).The numbers in the figure refer to each compound's K_(i) (in μM) forinhibition of [¹⁴C]Chrysamine G binding to the synthetic peptide, Aβ(10-43).

FIGS. 2A-2K illustrate the chemical structures of Chrysamine G andtri-alkyl tin derivatives of analogues of Chrysamine G, in particularheterocyclic analogues. Note that these structures represent one-half ofa molecule which is symmetric around the wavy bond shown in the upperright, except that the tri-alkyl tin moiety may only be on one side ofthe biphenyl group. The tri-alkyl tin derivatives are stableintermediate and immediate precursors for the preparation of highspecific activity halogenated radioactive derivatives. The heterocyclicanalogues represent alternative means of placing weakly acidic moietiesin the same structural position as the moderately acidic carboxylic acidgroup of Chrysamine G. These tri-alkyl tin precursor compounds are shownin their protonated form, yet those of skill in the art recognize thattheir deprotonated forms and tautomers also are embraced by thesedrawings.

2A) Chrysamine G.

2B) Tri-alkyl tin derivative of Chrysamine G;

2C) Tri-alkyl tin derivative of the 3-Hydroxy-1,2-benzisoxazoleanalogue;

2D) Tri-alkyl tin derivative of the phthalimide orisoindole-1,3(2H)-dione analogue;

2E) Tri-alkyl tin derivative of the phthalhydrazide or2,3-benzodiazine-1,4(2H,3H)-dione analogue;

2F) Tri-alkyl tin derivative of the 2,3-benzoxazine-1,4(3H)-dioneanalogue;

2G) Tri-alkyl tin derivative of the (2H)1,3-benzoxazine-2,4(3H)-dioneanalogue;

2H) Tri-alkyl tin derivative of the (3H)2-benzazine-1,3(2H)-dioneanalogue;

2I) Tri-alkyl tin derivative of the 1,8—Naphthalimide analogue.

2J) Tri-alkyl tin derivative of the tetrazole analogue.

2K) Tri-alkyl tin derivative of the oxadiazole analogue.

FIG. 3 shows displacement curves of [¹⁴C]Chrysamine G binding to Aβ(10-43) by several structural analogues of Chrysamine G. Abbreviationsrefer to those used in FIG. 1. FIG. 3A) Chrysamine G (open triangles);(5-FSA)CG (filled diamonds); 3,3′-(COOH)₂CG (filled squares);2,2′-(SO₃)₂CG (filled circles). FIG. 3B) Chrysamine G (open triangles);Congo red (open circles); aniline derivative (open inverted triangles);phenol derivative (open squares); salicylic acid (X's). Curves whichshow increased binding at higher concentrations do so because of theformation of micelles. Bedaux, F. et al., Pharm. Weekblad 98: 189(1963).

FIG. 4A is a Scatchard plot of Chrysamine G binding to Aβ (10-43). Thecurved line represents a nonlinear least-squares fit to a two,independent binding site model. The straight lines represent theindividual components.

FIG. 4B is a Scatchard analysis of [¹⁴C]CG binding to typical control(diamonds) and AD brain samples (squares). The dashed line has the sameslope as the AD line and is meant to aid in the comparison with thecontrol slope. This AD brain sample had 48 NP/×200 magnification, aK_(D) of 0.35 μM, and a B_(max) of 790 fmol/μg protein. The control hada K_(D) of 0.48 μM, and a B_(max) of 614 fmol/μg protein.

FIG. 5 is a graph illustrating the linearity of the binding assay withrespect to peptide concentration. Approximately 0.9 μg of Aβ (10-43) wasused in the typical assay.

FIG. 6A is a graph illustrating the time course of association ofChrysamine G and Aβ (10-43).

FIG. 6B is the graphic illustration of the determination of theassociation rate constant (k₁)

FIG. 6C is a graph of the time course of dissociation of Chrysamine Gfrom Aβ (10-43).

FIG. 7 is a graphic representation of a molecular model of theinteraction between Chrysamine G and Aβ.

FIG. 8A is a graph illustrating the correlation between the amount of[¹⁴C]Chrysamine G bound and the number of neuritic plaques (NP) in ADbrain samples.

FIG. 8B is a graph illustrating the correlation between the amount of[¹⁴C]Chrysamine G bound and the number of neurofibrillary tangles (NFT)in AD brain samples. In both FIG. 8A and 8B, the x-axis represents theaverage NP or NFT count per field at ×200 magnification in Bielschowskystained sections of either the superior/middle frontal (n=10) orsuperior temporal cortex (n=10). The filled symbols and heavy linesindicate brains without amyloid angiopathy, the open symbols and dashedlines indicate brains with amyloid angiopathy. The y-axis representstotal, absolute [¹⁴C]Chrysamine G binding (fmol/μg protein) inhomogenates of brain samples adjacent to those used for staining.Approximately 75 μg protein and 150 nM [¹⁴C]Chrysamine G were used.

FIGS. 9A, 9B and 9C. The binding of Chrysamine G to various brain areasin samples of AD brain having more than 20 NPs/×200 magnification,referred to as “High Plaque AD Brains”, is shown in FIG. 9A. The bindingof Chrysamine G to brain areas in samples of AD brain having less than20 NPs/×200 magnification, referred to as “Low Plaque AD Brains”, isshown in FIG. 9B. The data points represent the ratio of [¹⁴C]ChrysamineG binding in the designated brain area to [¹⁴C]Chrysamine G binding inthe cerebellum (CB) of the same brain. Horizontal bars represent themean and error bars represent the standard error for control (circles),and AD brain (diamonds in 9A and 9B). Brain areas include the frontalpole (FP), head of caudate (CAU), superior/middle frontal (SMF),superior temporal (ST), inferior parietal (IP), and occipital (OC)cortex. Asterisks indicate significant differences compared to control(*p<0.05; **p<0.001). Two Down's syndrome brain samples are indicated inFIG. 9C. The diamonds in 9C represent a brain from a 23 year old Down'ssyndrome patient not yet symptomatic with AD. The triangles in 9Crepresent a 51 year old Down's syndrome patient who had developed AD asdo the vast majority of Down's syndrome patients by their 40's.

FIG. 10 is a graph illustrating the tissue levels of Chrysamine G inmice injected with [¹⁴C]Chrysamine G in the lateral tail vein andsacrificed at the times indicated. The open symbols and thin linesrepresent absolute radioactivity in units of cpm/g tissue (left axis).The closed symbols and solid lines represent the ratio of brainradioactivity to that in kidney (top) or blood (middle). The ratios areplotted on the right axis.

FIG. 11. TOP: Section from the inferior temporal lobe of AD brainstained with Chrysamine G by the method of Stokes and Trickey, J. Clin.Pathol. 26: 241-242 (1973). Two neuritic plaques are clearly visible.BOTTOM: Adjacent sections from the temporal lobe of an AD patient withamyloid angiopathy. Left: Brain section was stained with the Congo redmethod of Puchtler. Puchtler et al., J. Histochem. Cytochem. 10: 35(1962). The bar represents 20 microns. Right: Adjacent brain sectionstained by substituting Chrysamine G for Congo red in the Puchtlermethod. Both sections readily demonstrate the same amyloid-laden vessel.A small amount of background autofluorescence from erythrocytes andlipofuscin also is visible. All photomicrographs were obtained usingfluorescein isothiocyanate (FITC) filters.

FIG. 12. A bar graph showing the effect of increasing concentrations ofAβ (25-35) in the presence and absence of Chrysamine G on the cellularredox activity of rat pheochromocytoma (PC-12) cells as measured by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, MTT,reduction. The reduction product of MTT absorbs at 560 nm which isplotted on the vertical axis. The effect of Aβ (25-35) alone is shown inthe filled bars and shows a dose dependent decrease in MTT reduction.Significant differences from control (no Aβ (25-35), no Chrysamine G)are shown in white inside the filled bars. The protective effect of 20μM Chrysamine G is shown in the open bars. Significant differencesbetween MTT reduction in the presence and absence of Chrysamine G areshown in black inside the open bars.

FIG. 13. A bar graph showing the protective effect of increasingconcentrations of Chrysamine G against the Aβ (25-35)-induced reductionof cellular redox activity of rat pheochromocytoma (PC-12) cells asmeasured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, MTT, reduction. The reduction product of MTT absorbs at 560 nmwhich is plotted on the vertical axis. The effect of Chrysamine G in theabsence of Aβ (25-35) is shown in the filled bars. There was nosignificant difference between control (no Aβ (25-35), no Chrysamine G)and any of the concentrations of Chrysamine G in the absence of Aβ(25-35). MTT reduction in the presence of 1 μM Aβ (25-35) and increasingconcentrations of Chrysamine G is shown in the open bars. Significantdifferences in MTT reduction between the presence and absence of Aβ(25-35) at each concentration of Chrysamine G are shown in white insidethe filled bars. Significant differences in MTT reduction between the Aβ(25-35) control (no Chrysamine G) and Aβ (25-35) plus increasingconcentrations of Chrysamine G are shown in black inside the open bars.

FIG. 14. Comparison of the effects of Chrysamine-G and the inactivephenol derivative on the toxicity induced by Aβ (25-35). 1 μM Aβ (25-35)was present in all experiments except control. Chrysamine-G showedprotective effects at 0.1 and 1 μM, but the phenol derivative showed noprotective effects, and perhaps enhanced the toxicity of Aβ.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention exploits the ability of Chrysamine G andradiolabeled derivatives thereof to cross the blood brain barrier invivo and bind to Aβ deposited in neuritic (but not diffuse) plaques, toAβ deposited in cerebrovascular amyloid, and to the amyloid consistingof the protein deposited in NFT. Chrysamine G is a Congo red derivativewith the key structural difference being that the sulfonic acid moietiesfound in Congo red are replaced by carboxylic acid groups in ChrysamineG (FIG. 1). This structural alteration allows Chrysamine G to enter thebrain better than Congo red and large macromolecules such as antibodies.Tubis et al., J. Am. Pharmaceut. Assn. 49: 422 (1960). Also, ChrysamineG may be a more specific marker for AD pathophysiology than antibodies,which would also label diffuse plaques, due to the apparent specificityof Chrysamine G for the neuritic plaque and NFTs.

The Chrysamine G derivatives of the present invention have each of thefollowing characteristics:

(1) specific binding to synthetic Aβ in vitro, (2) binding to neuriticbut not diffuse plaques in brain sections (3) ability to cross anon-compromised blood brain barrier in vivo.

The method of this invention determines the presence and location ofamyloid deposits in an organ or body area, preferably brain, of apatient. The present method comprises administration of a detectablequantity of a pharmaceutical composition containing a compound ofFormula I, as defined above, called a “detectable compound,” or apharmaceutically acceptable water-soluble salt thereof, to a patient. A“detectable quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable detection of binding of thecompound to amyloid. An “imaging effective quantity” means that theamount of the detectable compound that is administered is sufficient toenable imaging of binding of the compound to amyloid.

The invention employs amyloid probes which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The term “invivo imaging” refers to any method which permits the detection of alabeled Chrysamine G, labeled Chrysamine G derivative or labeledcompound of Formula I, as described above, of the present invention. Forgamma imaging, the radiation emitted from the organ or area beingexamined is measured and expressed either as total binding or as a ratioin which total binding in one tissue is normalized to (for example,divided by) the total binding in another tissue of the same subjectduring the same in vivo imaging procedure. Total binding in vivo isdefined as the entire signal detected in a tissue by an in vivo imagingtechnique without the need for correction by a second injection of anidentical quantity of labeled compound along with a large excess ofunlabeled, but otherwise chemically identical compound. A “subject” is amammal, preferably a human, and most preferably a human suspected ofhaving dementia.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁹F are particularly suitable for in vivoimaging in the methods of the present invention. The type of instrumentused will guide the selection of the radionuclide or stable isotope. Forinstance, the radionuclide chosen must have a type of decay detectableby a given type of instrument. Another consideration relates to thehalf-life of the radionuclide. The half-life should be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that the host does not sustain deleteriousradiation. The radiolabeled compounds of the invention can be detectedusing gamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradiolabel will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

In the present invention, amyloid binding compounds/probes are madewhich are useful for in vivo imaging and quantification of amyloiddeposition. These compounds are to be used in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the Chrysamine G derivatives may be labeled with¹⁹F or ¹³C for MRS/MRI by general organic chemistry techniques known tothe art. See, e.g., March, J. “ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985), the contents of which arehereby incorporated by reference. The Chrysamine G derivatives also maybe radiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contentsof which are hereby incorporated by reference. The Chrysamine Gderivatives also may be radiolabeled with ¹²³I for SPECT by any ofseveral techniques known to the art. See, e.g., Kulkarni, Int. J. Rad.Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are herebyincorporated by reference. In addition, the Chrysamine G derivatives maybe labeled with any suitable radioactive iodine isotope, such as, butnot limited to ¹³¹I, ¹²⁵I, or ¹²³I, by iodination of a diazotized aminoderivative directly via a diazonium iodide, see Greenbaum, F. Am. J.Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amineto the stable triazene, or by conversion of a non-radioactivehalogenated precursor to a stable tri-alkyl tin derivative which thencan be converted to the iodo compound by several methods well known tothe art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983),Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J.Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med.Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994);Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stabletriazene or tri-alkyl tin derivative of Chrysamine G or its analogues isreacted with a halogenating agent containing ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable tri-alkyl tin derivatives ofChrysamine G and its analogues are novel precursors useful for thesynthesis of many of the radiolabeled compounds within the presentinvention. As such, these tri-alkyl tin derivatives are one embodimentof this invention.

The Chrysamine G derivatives also may be radiolabeled with known metalradiolabels, such as Technetium-99m (^(99m)Tc). Modification of thesubstituents to introduce ligands that bind such metal ions can beeffected without undue experimentation by one of ordinary skill in theradiolabeling art. The metal radiolabeled Chrysamine G derivative canthen be used to detect amyloid deposits.

The methods of the present invention may use isotopes detectable bynuclear magnetic resonance spectroscopy for purposes of in vivo imagingand spectroscopy. Elements particularly useful in magnetic resonancespectroscopy include ¹⁹F and ¹³C.

Suitable radioisotopes for purposes of this invention includebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.

Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁹F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.The preferred radiolabels are ¹⁸F for use in PET in vivo imaging, ¹²³Ifor use in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C f or in vitrostudies. However, any conventional method for visualizing diagnosticprobes can be utilized in accordance with this invention.

The method could be used to diagnose AD in mild or clinically confusingcases. This technique would also allow longitudinal studies of amyloiddeposition in human populations at high risk for amyloid deposition suchas Down's syndrome, familial AD, and homozygotes for the apolipoproteinE4 allele. Corder et al., Science 261: 921 (1993). A method that allowsthe temporal sequence of amyloid deposition to be followed can determineif deposition occurs long before dementia begins or if deposition isunrelated to dementia. This method can be used to monitor theeffectiveness of therapies targeted at preventing amyloid deposition.

Generally, the dosage of the detectably labeled Chrysamine G derivativewill vary depending on considerations such as age, condition, sex, andextent of disease in the patient, contraindications, if any, concomitanttherapies and other variables, to be adjusted by a physician skilled inthe art. Dosage can vary from 0.001 mg/kg to 1000 mg/kg, preferably 0.1mg/kg to 100 mg/kg.

Administration to the subject may be local or systemic and accomplishedintravenously, intraarterially, intrathecally (via the spinal fluid) orthe like. Administration may also be intradermal or intracavitary,depending upon the body site under examination. After a sufficient timehas elapsed for the compound to bind with the amyloid, for example 30minutes to 48 hours, the area of the subject under investigation isexamined by routine imaging techniques such as MRS/MRI, SPECT, planarscintillation imaging, PET, and emerging imaging techniques, as well.The exact protocol will necessarily vary depending upon factors specificto the patient, as noted above, and depending upon the body site underexamination, method of administration and type of label used; thedetermination of specific procedures would be routine to the skilledartisan. For brain imaging, preferably, the amount (total or specificbinding) of the bound radioactively labelled Chrysamine G or ChrysamineG derivative or analogue is measured and compared (as a ratio) with theamount of labelled Chrysamine G or Chrysamine G derivative bound to thecerebellum of the patient. This ratio is then compared to the same ratioin age-matched normal brain.

The pharmaceutical compositions of the present invention areadvantageously administered in the form of injectable compositions. Atypical composition for such purpose comprises a pharmaceuticallyacceptable carrier. For instance, the composition may contain about 10mg of human serum albumin and from about 0.5 to 500 micrograms of thelabeled Chrysamine G or Chrysamine G derivative per milliliter ofphosphate buffer containing NaCl. Other pharmaceutically acceptablecarriers include aqueous solutions, non-toxic excipients, includingsalts, preservatives, buffers and the like, as described, for instance,in REMINGTON'S PHARMACEUTICAL SCIENCES, 15th Ed. Easton: Mack PublishingCo. pp. 1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV.,14th Ed. Washington: American Pharmaceutical Association (1975), thecontents of which are hereby incorporated by reference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobials, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components of the pharmaceutical composition are adjustedaccording to routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

Particularly preferred pharmaceutical compositions of the presentinvention are those that, in addition to specifically binding amyloid invivo and capable of crossing the blood brain barrier, are also non-toxicat appropriate dosage levels and have a satisfactory duration of effect.

Molecular Modeling

Molecular modeling was done on an Evans and Sutherland PS-330 computergraphics system, running the computer modeling program MacroModel(Version 2.5 available from C. Still at Columbia University) to generatethe AD peptide chains in the anti-parallel beta-sheet conformation.Kirschner et al., Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986). Theamyloid peptides were used without further structural refinement. The Aβpeptides were aligned so that alternate chains were spaced 4.76 Å apart,characteristic of beta-sheet fibrils. Kirschner, supra. Chrysamine G wasenergy minimized and aligned with the fibril model to maximize contactwith lysine-16 of Aβ (10-43) and the hydrophobic phenylalanine-19 and-20 region.

Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity,Kinetics, Maximum Binding

The characteristics of Chrysamine G and Chrysamine G derivative bindingare first analyzed using synthetic Aβ peptide called Aβ (10-43). The10-43 peptide was chosen because it has been shown that this peptideprovides a model system containing all of the characteristic structuralfeatures of Aβ peptides. Hilbich et al., J. Mol. Biol. 218: 149 (1991).The 10-43 amino acid fragment of Aβ was synthesized with9-fluorenylmethyl chloroformate (FMOC) chemistry by the PeptideSynthesis Facility of the University of Pittsburgh. The peptide wascharacterized by mass spectrometry and the major component had an M_(R)of 3600 g/mole (calc. 3598). The peptide was further purified by themethod of Hilbich et al. which, in brief, consisted of sequentialsize-exclusion chromatography on a Biogel P10 column (2×180 cm, 200-400mesh, Biorad, Richmond, Calif.) in 70% formic acid followed by a secondelution through a Biogel P4 column (2×180 cm, 200-400 mesh) in 1M aceticacid. Hilbich et al., J. Mol. Biol. 218: 149 (1991). The peptide waslyophilized and stored at −80° C. until used in the binding assays.

Amino acid sequence for Aβ (10-43) is as Follows:

10 11 12 13 14 15 16 17 18 19 20 21 Tyr Glu Val His His Gln Lys Leu ValPhe Phe Ala 22 23 24 25 26 27 28 29 30 31 32 33 Glu Asp Val Gly Ser AsnLys Gly Ala Ile Ile Gly 34 35 36 37 38 39 40 41 42 43 Leu Met Val GlyGly Val Val Ile Ala Thr

Binding assay to synthetic Aβ (10-43)

Binding assays were performed in 12×75 mm borosilicate glass tubes.Various concentrations of nonradioactive Chrysamine G derivatives wereadded in 10% ethanol/water. Ethanol was necessary to prevent the micelleformation which occurs with these diazo dye derivatives, since themicelles are trapped by the filter even in the absence of peptide. Tothe above solution, 25 μl of a 0.36 mg/ml suspension of Aβ (10-43) inH₂O was added and 10% ethanol was added to bring the volume to 950 μl.After incubating for 10 min at room temperature, 50 μl of[¹⁴C]Chrysamine G in 40% ethanol was added, resulting in a finalconcentration of [¹⁴C]Chrysamine G of 100-125 nM depending on thepreparation of [¹⁴C]Chrysamine G used. The binding mixture was incubatedfor 30 min at room temperature. Bound and free radioactivity wereseparated by vacuum filtration through Whatman GF/B filters using aBrandel M-24R Cell Harvester (Gaithersburg, Md.) followed by two 3-mlwashes with 10% ethanol at room temperature. Filters were equilibratedovernight in 4 ml Cytoscint®-ES scintillant (ICN Biomedicals, Inc.,Irvine, Calif.) in 7.0 ml plastic scintillation vials before counting.In this and all binding assays, incubations were done at least intriplicate and the results expressed as mean±standard deviation.

Kinetic Studies

Kinetics studies of [¹⁴C]Chrysamine G binding to Aβ (10-43) wereperformed in 13×100 mm borosilicate glass tubes by the filtration assaydescribed above. For the kinetics of association, 25 μl of 0.36 mg/ml Aβ(10-43) were placed in 475 μl of 10% ethanol and 4.5 ml of 125 nM[¹⁴C]Chrysamine G was added to the solution at time zero. The mixturewas rapidly vortexed and the binding reaction was stopped by vacuumfiltration through Whatman GF/B filters using a Brandel M-24R CellHarvester (Gaithersburg, Md.) followed by two 3-ml washes with 10%ethanol at room temperature at times of 5, 10, 20, 30, 45, 60, 75, 135,240, and 300 sec.; bound radioactivity was determined as above.

For the kinetics of dissociation, 25 μl of 0.36 mg/ml Aβ (10-43) wereplaced in 450 μl of 10% ethanol followed by 25 μl of 2.5 μM[¹⁴C]Chrysamine G in 40% ethanol. This mixture was vortexed andincubated for 30 min at room temperature. The mixture was diluted with4.5 ml of 10 μM nonradioactive Chrysamine G in 10% ethanol at time zero,the mixture was rapidly vortexed, and the dissociation was stopped byfiltration as above at times of 0.5, 1.5, 3, 5, and 15 min, and boundradioactivity was determined as above.

Characterization of Specific Binding to Alzheimer's Disease Brain

Binding of Chrysamine G to AD and Control Brain Homogenates

Autopsy brain samples were obtained from the Neuropathology Core of theAlzheimer's Disease Research Center of the University of Pittsburgh.Controls were defined as not meeting neuropathological criteria for AD(sufficient number of NPs or NFTs) according to the standards specifiedin a published NIA conference report. Khachaturian, Arch. Neurol. 42:1097 (1985). Brain samples from eight control (ages 58-75), eleven AD(ages 61-84), and two Down syndrome brains (ages 23 and 51) werestudied. There were six high-plaque (>20 NPs/×200 magnification) andfive low-plaque (<20 NPs/×200 magnification) AD brains. Two controlswere clinically demented but had no NPs or NFTs and received thediagnosis of “Dementia Lacking Distinctive Histology”. Knopman Dementia4: 132 (1993). Another control had dementia and olivopontocerebellaratrophy. The other controls had no clinical or histological evidence ofneurologic disease. Autopsy samples were immediately frozen at −70° C.and stored at that temperature until homogenized. The numbers of NPs andNFTs were counted in sections of five separate but adjacent fields (×200magnification) between cortical layers 2 and 4 in the cortex at thejunction of superior and middle frontal gyri and superior temporalisocortex of all brains studied. A qualitative assessment of thepresence of amyloid angiopathy in the superior/middle frontal cortex wasmade. The Bielschowsky silver impregnation method was used to identifyNPs and NFTs and Congo red staining was used to identify cerebralamyloid angiopathy. Details of this procedure have been previouslypublished. Moossy et al., Arch. Neurol. 45: 251 (1988). Samples used forCG binding to the superior/middle frontal or superior temporal cortexwere adjacent on gross dissection to those used for NP and NFT counts.

Approximately 100 mg of tissue from the junction of the superior andmiddle frontal cortex, superior temporal cortex, frontal pole, head ofthe caudate, inferior parietal cortex, occipital cortex, or cerebellumwere homogenized with a Polytron® tissue homogenizer (PT 10/35, BrinkmanInstruments Inc., Westbury, N.Y.) for 30 sec at setting 6 in 10% ethanolat a concentration of 10-20 mg brain/ml. Not all areas were availablefrom each brain. Aliquots of 25-150 μl tissue (about 25-300 μg ofprotein by the method of Lowry et al., J. Biol, Chem. 193: 265 (1951))were incubated in 12×75 mm borosilicate glass tubes at room temperaturewith 10-750 nM [¹⁴C]CG (26.8 Ci/mole) in a final volume of 1.0 ml of 10%ethanol for 30 min at room temperature. The standard conditions employedabout 150 μg of protein and 75 nM [¹⁴C]CG for the cerebellar ratiostudies and about 75 μg of protein and 150 nM [¹⁴C]CG for thecorrelative studies with NPs, NFTS, and amyloid angiopathy. Ethanol wasnecessary to prevent the micelle formation which occurs with diazo dyederivatives, since the micelles are trapped by the filter even in theabsence of tissue. Bound and free radioactivity were separated by vacuumfiltration through Whatman GF/B filters using a Brandel M-24R CellHarvester (Gaithersburg, Md.) followed by two 3-ml washes with 10%ethanol at room temperature. Filters were equilibrated overnight in 4 mlCytoscint®-ES scintillant (ICN Biomedicals, Inc., Irvine, Calif.) in 7.0ml plastic scintillation vials before counting. Saturable (specific)binding was defined as total binding minus residual (non-saturable)binding in the presence of 20 μM unlabelled CG. In all binding assays,incubations were done at least in triplicate and the results expressedas mean±standard error unless otherwise specified. Results wereexpressed either in absolute terms of fmol [¹⁴C]CG bound/μg protein in agiven brain area, or as a ratio of the fmol/μg protein in that brainarea to the fmol/μg protein in the cerebellum of the same brain.

Octanol/Water Partitioning

Approximately 75 μM solutions of Chrysamine G or its analogues wereprepared in 5.0 ml 1-octanol. Five ml of phosphate buffered saline (0.15M NaCl, 5 mM potassium phosphate, pH 7.4) were added and the layersmixed by rapid vortexing. The mixture was then centrifuged at 1,000 g tofacilitate the formation of two clear phases. The layers were separatedusing a separatory funnel and 600 μl of each layer was diluted with 400μl of ethanol and the absorbance measured at 389 nm for Chrysamine G orthe λ_(max) for each analogue. Concentrations were determined aftercorrection for the molar absorptivity differences in the two solventsand the partition coefficient expressed as the concentration in theoctanol layer divided by the concentration in the aqueous layer.Experiments were done in triplicate.

Imaging the Binding of Chrysamine G to Amyloid Deposits in Alzheimer'sDisease Brain

For visual demonstration of CG binding to tissue, adjacent 8 micronparaffin sections of an AD brain with heavy deposits of cerebrovascularamyloid were stained with both CG and Congo red by the alkaline Congored method of Puchtler et al., J. Histochem. Cytochem. 10: 355 (1962) orthe method of Stokes and Trickey, J. Clin. Pathol. 26: 241-242 (1973).In the CG staining procedure, CG was substituted for Congo red, but theprocedure was otherwise identical. Stained slides were examined using afluorescein isothiocyanate (FITC) filter.

Determining Compound's Ability to Cross the Blood Brain Barrier

Mouse studies

Female Swiss-Webster mice were injected in the lateral tail vein withapproximately 0.03 μCi/g of [¹⁴C]Chrysamine G in a 0.9% NaCl solution.Mice were sacrificed by cervical dislocation at intervals of 15 min, 35min, 1 hr, 4 hr, and 24 hr after injection. The carotid blood, brain,liver, and kidneys were rapidly obtained, weighed, and homogenized indistilled/deionized H₂O using a ground glass homogenizer. An aliquot wasweighed into an 18.0 ml plastic scintillation vial (Beckman Poly-Q-Vial)and counted after addition of 10.0 ml of scintillation cocktail(Cytoscint®-ES (ICN)) and overnight equilibration. The [¹⁴C]Chrysamine Gcontent of the tissues was expressed as cpm/mg tissue.

Experiments in which radioactivity was extracted from tissues wereperformed as above except 0.05 μCi/g of [¹⁴C]Chrysamine G was injectedand the mice were sacrificed at 60 min. Brain and liver were thenremoved and extracted with a Folch procedure. Folch et al., J. Biol.Chem. 226: 447 (1957). In both tissues, over 95% of the extractedradioactivity was contained in the organic layer. The organic layer wasevaporated to dryness, resuspended in a minimal amount of 10%methanol/90% ACN, and injected onto a silica column (Prep Nova Pak HRSilica, 7.8×300 mm, Waters, Milford, Mass.) and eluted isocraticallywith the same solvent. Under these conditions, 99% of the radioactivityis eluted in the solvent front, but most lipids are retained longer,making the fraction eluting in the solvent front suitable for injectiononto the reverse-phase C4 column system described above. The entiresolvent front was collected, dried, and resuspended in 10% ACN/90%sodium phosphate buffer (5 mM, pH 6) and injected, along with authenticnon-radioactive Chrysamine G, onto the C4 column. One minute fractionswere collected and counted after addition of 10 ml of Cytoscint®-ES.

In yet another embodiment, the invention relates to a pharmaceuticalcomposition and method for preventing cell degeneration and toxicityassociated with fibril formation in certain “amyloidosis associated”conditions such as Alzheimer's Disease, Down's Syndrome and Type 2diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch),amyloid A (reactive), secondary amyloidosis, familial mediterraneanfever, familial amyloid nephropathy with urticaria and deafness(Muckle-wells Syndrome), amyloid lambda L-chain or amyloid kappa L-chain(idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, (systemic senile amyloidosises), AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele. This method involves administering apharmaceutical composition comprising Chrysamine G, or one of the abovedescribed derivatives thereof, to a subject suspected of having or athigh risk of developing such amyloidosis associated condition.

Because certain diazo compounds could be carcinogenic, the therapeuticcompounds of the present invention include only non-toxic,non-carcinogenic compounds. That is, the present invention addresses theproblems with potential carcinogenicity by using only diazo compoundsbased on 3,3′, 5,5′-tetramethylbenzidine. 3,3′,5,5′-Tetramethylbenzidine is a well-studied non-mutagenic,non-carcinogenic substitute for benzidine. Holland et al.. Tetrahedron,30: 3299-3302, (1974). De Serres and Ashby (eds.) Evaluation ofShort-Term Tests for Carcinogens, Elsevier North-Holland, 1981.3,3′,5,5′-Tetramethylbenzidine has been used as a substitute forbenzidine in the synthesis of azo dyes. Josephy and Weerasooriya,Chem.-Biol. Interactions, 1984: 375-382, (1984). Ashby et al.,Carcinogenesis 3: 1277-1282, (1982). Inventors have synthesized thetetramethyl analog of Chrysamine G(4,4′-bis(3-carboxy-4-hydroxyphenylazo)-3,3′,5,5′-tetramethylbiphenyl)and have shown it to bind to synthetic Aβ (10-43) with a Ki of 0.75±0.09μM, a value about twice that of Chrysamine G.

Any potential problems with lower bioavailability is avoided by the useof alkenyl and alkynyl derivatives of the azo compounds. These compoundsare not substrates for reduction by bacterial or mammalian azoreductases.

Indeed, compounds of the present invention intended for therapeutic useare advantageous over existing compounds because they contain either anon-mutagenic, non-carcinogenic benzidine derivative or an alkenyl oralkynyl linkage which is not a substrate for bacterial azo-reductases inthe intestines.

Tn vitro studies have shown that Aβ neurotoxicity requires fibrilformation and is inhibited by Congo red. Specifically, it has been shownthat the amyloid fibril-binding dye Congo red inhibits fibrillar Aβneurotoxicity by inhibiting fibril formation or by binding preformedfibrils. Lorenzo et al., Proc. Natl. Acad. Sci. USA 91: 12243-12247(1994). Congo red also has been shown to inhibit pancreatic islet celltoxicity of diabetes-associated amylin, another type of amyloid fibril.Lorenzo et al., supra. See also, Burgevin et al. NeuroReport 5: 2429(1994); Pollack et al., J. Neurosci. Letters 184: 113-116 (1995);Pollack et al. Neuroscience Letters 197: 211 (1995). These data indicatethat amyloid-binding compounds such as Chrysamine G and its derivatives,which are similar to Congo red but which, unlike Congo red, enter thebrain well, would be effective in preventing cell degeneration andtoxicity associated with fibril formation in amyloidosis associatedconditions.

In Example 8 and FIGS. 12 and 13, it is shown that Chrysamine G haseffects very similar to those previously reported for Congo red inhaving a dose-dependent, protective effect in rat pheochromocytoma.Therefore, these in vitro assays provide a means for selecting compoundsfor use in pharmaceutical compositions for the prevention of celldegeneration and toxicity associated with fibril formation.

Compounds such a Chrysamine G and the above described derivativesthereof, are tested pursuant to the present invention, for in vivoefficacy in preventing amyloid fibril formation or associated cellulardegeneration, as measured by the formation of dystrophic neurites,synapse loss, neurofibrillary tangle formation and gliosis, in an animalmodel, such as the “senile animal” model for cerebral amyloidosis,Wisniewski et al., J. Neuropathol. & Exp. Neurol. 32: 566 (1973), themouse model of familial Mediterranean fever (Neurochem., Inc. Kingston,Ontario, Canada) and the transgenic mouse model of Alzheimer-typeneuropathology, Games et al., Nature 373: 523-527 (1995). In thefamilial Mediterranean fever model, the animals develop systemicamyloidosis. In an in vivo assay according to this invention, serialnecropsies in animals treated and untreated with the compounds of thepresent invention to evaluate the inhibition of amyloid formation arecompared. In the animal models for cerebral amyloid formation, inaddition to following amyloid formation serially, the presence ofamyloid-associated neurodegeneration, as measured by the formation ofdystrophic neurites, synapse loss, neurofibrillary tangle formation andgliosis, also is assessed in serial necropsies in animals treated anduntreated with the compounds of the present invention.

According to the present invention, a pharmaceutical compositioncomprising Chrysamine G or derivatives thereof, is administered tosubjects in whom amyloid or amyloid fibril formation, cell degenerationand toxicity are anticipated. In the preferred embodiment, such subjectis a human and includes, for instance, those who are at risk ofdeveloping cerebral amyloid, including the elderly, nondementedpopulation and patients having amyloidosis associated diseases and Type2 diabetes mellitus. The term “preventing” is intended to include theamelioration of cell degeneration and toxicity associated with fibrilformation. By “amelioration” is meant the prevention of more severeforms of cell degeneration and toxicity in patients already manifestingsigns of toxicity, such as dementia.

The pharmaceutical composition for purposes of preventing celldegeneration and toxicity associated with fibril formation inamyloidosis associated diseases comprises Chrysamine G or a derivativethereof described above and a pharmaceutically acceptable carrier. Inone embodiment, such pharmaceutical composition comprises serum albumin,Chrysamine G or Chrysamine G derivative and a phosphate buffercontaining NaCl. Other pharmaceutically acceptable carriers includeaqueous solutions, non-toxic excipients, including salts, preservatives,buffers and the like, as described, for instance, in REMINGTON'SPHARMACEUTICAL SCIENCES, 15th Ed., Easton: Mack Publishing Co., pp.1405-1412 and 1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed.Washington: American Pharmaceutical Association (1975), and the UNITEDSTATES PHARMACOPEIA XVIII. 18th Ed. Washington: American PharmaceuticalAssociation (1995), the contents of which are hereby incorporated byreference.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

According to the invention, such pharmaceutical composition could beadministered orally, in the form of a liquid or solid, or injectedintravenously or intramuscularly, in the form of a suspension orsolution. By the term “pharmaceutically effective amount” is meant anamount that prevents cell degeneration and toxicity associated withfibril formation. Such amount would necessarily vary depending upon theage, weight and condition of the patient and would be adjusted by thoseof ordinary skill in the art according to well-known protocols. In oneembodiment, a dosage would be between 0.1 and 100 mg/kg per day, ordivided into smaller dosages to be administered two to four times perday. Such a regimen would be continued on a daily basis for the life ofthe patient. Alternatively, the pharmaceutical composition could beadministered intramuscularly in doses of 0.1 to 100 mg/kg every one tosix weeks.

In yet another embodiment, the invention relates to a method ofdetecting amyloid deposits in biopsy or post-mortem tissue. The methodinvolves incubating formalin-fixed tissue with a solution of a compoundof Formula I, described above. Preferably, the solution is 25-100%ethanol, (with the remainder being water) saturated with the compound ofFormula I. Upon incubation, the compound stains or labels the amyloiddeposit in the tissue, and the stained or labelled deposit can bedetected or visualized by any standard method. Such detection meansinclude microscopic techniques such as bright-field, fluorescence,laser-confocal and cross-polarization microscopy.

In yet another embodiment, the invention relates to a method ofquantifying the amount of amyloid in biopsy or post-mortem tissue. Thismethod involves incubating a labelled derivative of Chrysamine G,preferably the compounds of Formula I, or a water-soluble, non-toxicsalt thereof, with homogenate of biopsy or post-mortem tissue. Thetissue is obtained and homogenized by methods well known in the art. Thepreferred label is a radiolabel, although other labels such as enzymes,chemiluminescent and immunofluorescent compounds are well known toskilled artisans. The preferred radiolabel is ¹²⁵I, ¹⁴C or ³H, thepreferred label substituent of Formula I is at least one of R₁-R₇,R₁₀-R₄₉. Tissue containing amyloid deposits will bind to the labeledderivatives of Chrysamine G. The bound tissue is then separated from theunbound tissue by any mechanism known to the skilled artisan, such asfiltering. The bound tissue can then be quantified through any meansknown to the skilled artisan. See Example 3. The units of tissue-boundradiolabeled Chrysamine G derivative are then converted to units ofmicrograms of amyloid per 100 mg of tissue by comparison to a standardcurve generated by incubating known amounts of amyloid with theradiolabeled Chrysamine G derivative.

In yet another embodiment, the invention relates to a method ofdistinguishing an Alzheimer's diseased brain from a normal braininvolving obtaining tissue from (i) the cerebellum and (ii) another areaof the same brain, other than the cerebellum, from normal subjects andfrom subjects suspected of having Alzheimer's disease. See Example 3.Such tissues are made into separate homogenates using methods well knownto the skilled artisan, and then are incubated with a radiolabeledChrysamine G derivative. The amount of tissue which binds to theradiolabeled Chrysamine G derivative is then calculated for each tissuetype (e.g. cerebellum, non-cerebellum, normal, abnormal) and the ratiofor the binding of non-cerebellum to cerebellum tissue is calculated fortissue from normal and for tissue from patients suspected of havingAlzheimer's disease. These ratios are then compared. If the ratio fromthe brain suspected of having Alzheimer's disease is above 90% so of theratios obtained from normal brains, the diagnosis of Alzheimer's diseaseis made. The normal ratios can be obtained from previously obtained data(see Table 3), or alternatively, can be recalculated at the same timethe suspected brain tissuse is studied.

EXAMPLE 1 The Synthesis of Chrysamine G and Derivatives ThereofSynthesis of Chrysamine G

The synthesis of Chrysamine G (i.e.,4,4′-bis(3-carboxy-4-hydroxyphenylazo)-biphenyl) requires the followingreaction steps. These reaction steps will be referred to as the“Chrysamine G Synthesis” general procedure. Benzidine.2HCl (28.9 mg,0.11 mmole, Sigma Chemical Company, St. Louis, Mo.) was added to 1.5 mlof 1:1 DMSO:distilled/deionized H₂O in a 50 cc round bottom flask. Eachof the reaction steps were carried out at 0° C. unless otherwisespecified. Twenty-nine μl of concentrated HCl were added, resulting in aclear solution after stirring. To the benzidine solution, a solution of15.5 mg (0.22 mmole) of NaNO₂ in 300 μl of 1:1 DMSO/H₂O was addeddrop-wise, resulting in a pH of about 2-3. The reaction mixture wasstirred for 45 min, and then to this tetra-azotized benzidine mixturewas added drop-wise over a 10 min period to 24.8 mg (0.18 mmole) ofmethyl salicylate (Aldrich) dissolved in 2.0 ml of 100% DMSO containing250 mg/ml Na₂CO₃ in suspension, keeping the pH about 10.5. The resultingmixture was stirred for 1 hr at 0° C., and then overnight at roomtemperature.

After this time, the pH was adjusted to about 7 and the mixture wasextracted with three 50 ml portions of chloroform. The combinedchloroform extracts were washed with three 50 ml portions of H₂O, andthen taken to dryness yielding the dimethyl ester of Chrysamine G (i.e.,4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl), which wasfurther purified by recrystallization from chloroform/hexane. The esterwas then hydrolysed by dissolution in about 100 ml of 1:1 ethanol:H₂Ocontaining four equivalents of NaOH and refluxed for three hours.Evaporation of the ethanol followed by lyophilization of the H₂O yieldedthe tetra-sodium salt of Chrysamine G. The free acid of Chrysamine G wasformed by dissolving the tetra-sodium salt in H₂O, washing once withchloroform to remove any unhydrolysed dimethyl ester, lowering the pH toabout 2 and extracting with three 50 ml portions of ethyl acetate. Thecombined ethyl acetate extracts were washed with three 50 ml portions ofH₂O and taken to dryness. Alternatively, hydrolysis was accomplished bydissolving the ester in THF and adding 15-20 molar equivalents of solidpotassium tert-butoxide at room temperature and stirring for 15 minutes.After acidification, the free acid was extracted into ethyl acetatewhich was then evaporated. The tetra-sodium salt was formed by additionof sodium methoxide to a suspension of the free acid in ethanol untilthe pH was approximately 9.5.

Under these conditions, there was no remaining methyl salicylate,salicylic acid, or benzidine, and only trace amounts of themono-substituted product,4-hydroxy-4′-(3-carboxy-4-hydroxyphenylazo)-biphenyl by reverse-phaseHPLC using a C4 column (Vydac 214-TP510) using a solvent system ofsodium phosphate buffer (5 mM, pH 6):acetonitrile (ACN) 90:10,isocratically, for 10 min and then increased to 50% ACN over the next 20min at a flow rate of 3.5 ml/min. The column eluant was monitored at 290and 365 nm with a dual wavelength, diode array detector (Perkin Elmer235C). Under these conditions, Chrysamine G eluted at 17.6 min.

The structure of Chrysamine G and derivatives was confirmed by protonNMR at 500 MHz in DMSO-d₆ with TMS as the internal standard. The peakassignments for the tetra-sodium salt of Chrysamine G were as followswith SA referring to protons at the specified ring position on thesalicylic acid moiety and BZ referring to protons on the benzidinemoiety: SA-3, doublet J=8.73 Hz at 6.75 parts per million (ppm); SA-4,doublet of doublets J=8.73 and 2.72 Hz at 7.82 ppm; BZ-2/6, doubletJ=8.44 Hz at 7.91 ppm; BZ-3/5, doublet J-8.44 Hz at 7.95 ppm; and SA-6,doublet J-2.72 Hz at 8.28 ppm. The UV/visible spectrum in 40% ethanolshowed a λ_(max) at 389 nm. The molar absorptivity of Chrysamine G wasdetermined by calculating the concentration of Chrysamine G throughcomparison of peak areas to an internal standard by NMR and thenimmediately running the UV/vis spectrum of an aliquot of the NMR samplediluted in 40% ethanol. The molar absorptivity in 40% ethanol at 389 nmwas 5.5×10⁴ AU/(cm.M).

[¹⁴C]Chrysamine G was synthesized by a modification of the aboveprocedure. The tetra-azotization of benzidine was performed as describedabove except in 100% H₂O. Fifty μl of 2.5 M Na₂CO₃ in H₂O were added to50 μCi of crystalline salicylic acid-carboxy-¹⁴C (Sigma) in a 0.5 mlconical glass vial. Sixty μl of the tetra-azotized benzidine mixture wasadded to the conical vial, vortexed and kept at 0° C. for 1 hr. Toprevent formation of the mono-substituted benzidine by-product, 12.5 μlof 250 mM non-radioactive salicylic acid (Sigma) in 2.5 M Na₂CO₃ wasadded to the reaction mixture and maintained for 1 hr at 0° C. The vialwas kept overnight at room temperature. The entire mixture was dissolvedin a minimal amount of 35% ACN and injected onto the C4 column asdescribed above. The peak corresponding to the Chrysamine G standard wascollected and lyophilized. A specific activity of 26.8 Ci/mole wascalculated by determining the absorbance at 389 nm and then counting theradioactivity in an aliquot of the same sample. The [¹⁴C]Chrysamine Gwas stored in 40% ethanol. When the purified [¹⁴C]Chrysamine G wasre-injected onto the C4 column and eluted isocratically with 21% ACN at3.5 ml/min, >98% of the radioactivity co-eluted with authenticChrysamine G at 10.4 min. Many of the Chrysamine G derivatives weresynthesized using this “Chrysamine G Synthesis” general procedure, withthe exceptions noted below. Structures of the derivatives were verifiedby NMR. FIG. 1 shows the chemical structure of Chrysamine G and severalderivatives.

[³H]Chrysamine G, or other [³H]Chrysamine G derivatives such as4,4′-bis(3-carboxy-4-hydroxy-5-phenylphenylazo)-3,3′-[³H]biphenyl wassynthesized using 3,3′-[³H]benzidine. The 3,3′-[³H]benzidine (specificactivity 50 Ci/mmole) was custom synthesized by American RadiolabeledChemicals (St. Louis, Mo.) by exposing 3,3′-diiodobenzidine (see below)to a large excess of high specific activity tritium gas in the presenceof any of several catalysts well known in the art and excessN,N-di-isopropyl-ethylamine to consume acid generated in the reduction.After removal of the labile tritium, preparative TLC was used toseparate the 3,3′-[³H]benzidine from a small amount of incompletelyreduced mono-iodobenzidine impurity. Specifically, 11 nmoles of3,3′-[³H]benzidine in 562 μl of 0.01 N HCl on ice, stirred in a glassvial, was treated at one minute intervals with five portions of 2.2μmoles of NaNO₂ in 5 μl H₂O. After the last portion, 22 μmoles of3-phenylsalicylic acid (TCI) in 250 μl of 2.5 M Na₂CO₃ was added and theice was removed. After 5 minutes 50μl of 5 N HCl is added followed by887 μl of ethanol. The4,4′-bis(3-carboxy-4-hydroxy-5-phenylphenylazo)-3,3′-[³H]biphenyl waspurified by reverse-phase HPLC using a C4 column (Vydac 214-TP510) usinga solvent system of sodium phosphate buffer (5 mM, pH 6.5)/ethanol (2.0ml/min; 40-50% ethanol over a 10 min concave gradient, then holding at50% for 15 min). The product co-eluted with authentic sample.

Synthesis of the 3-Isopropylsalicylic Acid Derivative of Chrysamine G

4,4′-bis(3-carboxy-4-hydroxy-5-isopropylphenylazo)-3-iodobiphenyl issynthesized by substituting 3-bromobenzidine (see below) for benzidineand methyl 3-isopropylsalicylate for methyl salicylate in the ChrysamineG Synthesis described above. The bromo derivative thus obtained isconverted to the tri-alkyl tin and then to the iodo derivative asdescribed in detail below.

Synthesis of the 3-Phenyl-, 3-(2-Phenylethene)-, and3-(2-Phenylethyl)-salicylic Acid Derivative of Chrysamine G

4,4′-bis(3-carboxy-4-hydroxy-5-phenylphenylazo)-3-iodobiphenyl issynthesized by substituting 3-bromobenzidine (see below) for benzidineand methyl 3-phenylsalicylate [made by esterification of3-phenylsalicylic acid (TCI America, Portland, Oreg.)] for methylsalicylate in the Chrysamine G Synthesis described above. The bromoderivative thus obtained is converted to the tri-alkyl tin and then tothe iodo derivative as described in detail below. Other derivativesusing benzidine or other substituted benzidines described below are madeby substituting the appropriate benzidine derivative.

4,4′-bis(3-carboxy-4-hydroxy-5-(2-phenylethene)-phenylazo)-3-iodobiphenylis made by the above procedure using methyl3-(2-phenylethene)-salicylate in the place of methyl salicylate.3-(2-phenylethene)-salicylate is synthesized by coupling diethylbenzylphosphonate (Aldrich) to 3-formylsalicylic acid (Aldrich) by theprocedure described below for the synthesis of vinyl (C═C) derivativesof Chrysamine G.

4,4′-bis(3-carboxy-4-hydroxy-5-(2-phenylethyl)-phenylazo)-3-iodobiphenylis made by the above procedure using methyl 3-(2-phenylethyl)-salicylatein the place of methyl salicylate. 3-(2-phenylethyl)-salicylate issynthesized by reduction of 3-(2-phenylethene)-salicylate with hydrogenover a palladium or platinum on carbon catalyst.

Synthesis of Vinyl (C═C) derivatives of Chrysamine-G

4,4′-biphenyldicarboxylic acid (Aldrich) is converted by reduction withLiAlH₄ to 4,4′-bis(hydroxymethyl)biphenyl, which, in turn, is convertedto 4,4′-bis(iodomethyl)biphenyl by reaction with NaI and BF₃-etherate inACN. The iodo compound is heated to 90° C. for one hour with excesstriethyl phosphite to produce tetraethyl4,4′-biphenyldimethylphosphonate. Similar treatment of1,4-napthalene-dicarboxylic acid (Aldrich) or9,10-anthracene-dicarboxylic acid (Aldrich) yields the respectivetetraethyl phoshonates. After recrystallization from hexane, thephosphonate is dissolved in DMF and treated with a ten-fold excess ofsodium methoxide, followed by two equivalents of 5-formylsalycylic acidin DMF. After stirring at room temperature for 24 hrs, the reactionmixture is poured into water. Acidification of the water to pH 5.0 withHCl causes precipitation of the flourescent product,4,4′-bis(3-carboxy-4-hydroxyphenylethenyl)-biphenyl, which can beselectively extracted into ethyl acetate from any mono-substitutedby-product. Similar treatment of tetraethyl p-xylylenediphosphonate (TCIAmerica) with 5-formylsalicylic acid, or it's derivatives, gives1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-benzene. Similar treatment ofdiethyl benzylphosphonate (Aldrich) with 3-formylsalicylic acid, gives3-(2-phenylethene)-salicylic acid. Likewise1,4-bis(3-carboxy-4-hydroxyphenylethenyl)-naphthalene or9,10-bis(3-carboxy-4-hydroxyphenylethenyl)-anthracene is obtained bytreating the appropriate phosphonate with 5-formylsalicylic acid. Otherderivatives are obtained by the use of other formylsalicylic acidcongeners, formyl benzoic acids, or hydroxy- or methoxybenzaldehydes.

Synthesis of Amide derivatives of Chrysamine G

4,4′-biphenyldicarboxylic acid (Aldrich) is converted to the acidchloride by reaction with excess thionyl chloride in DMF. After removalof the remaining thionyl chloride on a rotary evaporator, this acidchloride is added to a solution of two equivlents of 5-aminosalicylicacid and triethylamine. The4,4′-bis(3-carboxy-4-hydroxyphenylamido)-biphenyl precipitates uponpouring the DMF solution into dilute HCl.

Synthesis of Schiff Base (CH═N) derivatives of Chrysamine G

4,4¹-biphenyldicarboxylic acid (Aldrich) is converted by reduction withLiAlH₄ to 4,4′-bis(hydroxymethyl)biphenyl, which, in turn, is convertedto 4,4′-biphenyldicarboxaldehyde by treatment with BaMnO₄ in ethylacetate. The dialdehyde is dissolved in DMF and treated with5-aminosalicylic acid dissolved in DMF. The resultant suspension ofSchiff base is added to ten volumes of water and extracted into ethylacetate to yield the desired product. Alternatively, 3- or5-formylsalicylic acid can be coupled in an analogous manner tosubstituted or unsubstituted benzidine analogues.

Synthesis of Amine derivatives of Chrysamine G

The suspension of Schiff base in DMF described above is treated withexcess NaBH₄ dissolved in ethanol and then refluxed for one hr. Theresulting secondary amine is isolated by neutralizing the excess NaBH₄with HCl, pouring the solution into ten volumes of water, and extractinginto ethyl acetate.

Synthesis of Hydrazo (NH—NH) Derivatives of Chrysamine G

Chrysamine G and other azo compounds are dissolved in ethanol andtreated with two equivalents of PdCl₂ and ten equivalents of NaBH₄ atroom temperature. The solution qiuckly looses its color. The ethanolsuspension is filtered to removed the reduced palladium, poured intodilute HCl, and extracted into ethyl acetate.

Synthesis of Hydrazone (NH—N═C) Derivatives

In general, hydrazone derivatives are prepared by reaction of4,4′-dihydrazinobiphenyl with a suitable ketone, or by reaction ofsubstituted or unsubstituted tetra-azotized benzidine derivatives withactive hydrogen compounds. A specific example of the latter methodfollows. Benzidine is tetra-azotized by the same procedure used for thesynthesis of Chrysamine G. This tetra-azotized solution is added to asolution of two equivalents of4-hydroxy-5-methyl-4-cyclopentene-1,3-dione (Aldrich) dissolved in DMSOcontaining 250 mg/ml Na₂CO₃ in suspension, keeping the pH about 10.5.The resulting mixture was stirred for 1 hr at 0° C., and then overnightat room temperature. The hydrazone product was obtained by dilution ofthe reaction mixture into ten volumes of water, acidification with HCl,and extraction into ethyl acetate.

Synthesis of Pyridine and Diazine Derivatives of Chrysamine G

4,4′-bis(5-carboxy-6-hydroxy-3-pyridylazo)-3-iodobiphenyl and relatedhydroxypyridinecarboxylic or hydroxydiazinecarboxylic acid derivativesof Chrysamine G are synthesized by substituting 3-bromobenzidine (seebelow) for benzidine and 2-hydroxynicotinic acid (or, for relatedderivatives, 6-hydroxynicotinic acid, 3-hydroxypicolinic acid, orhydroxydiazinecarboxylic acids such as 4-hydroxypyrimidine-6-carboxylicacid) for methyl salicylate in the Chrysamine G Synthesis describedabove. The bromo derivative thus obtained is converted to the tri-alkyltin and then to the iodo derivative as described in detail below.

Synthesis of Naphthalene, Quinoline, and Benzodiazine Derivatives ofChrysamine G

4,4′-bis(3-carboxy-4-hydroxy-naphthylazo)-3-iodobiphenyl and relatedhydroxynaphthoic, hydroxyquinolinecarboxylic, orhydroxybenzodiazinecarboxylic acid derivatives of Chrysamine G aresynthesized by substituting 3-bromobenzidine (see below) for benzidineand 1-hydroxy-2-naphthoic acid (or, for related derivatives, otherhydroxynaphthoic acid isomers, hydroxyquinolinecarboxylic acids such askynurenic acid, or hydroxybenzodiazinecarboxylic acids such as5-hydroxyquinoxaline-6-carboxylic acid) for methyl salicylate in theChrysamine G Synthesis described above. The bromo derivative thusobtained is converted to the tri-alkyl tin and then to the iododerivative as described in detail below.

Synthesis of Nitrophenol Derivatives of Chrysamine G

4,4′-bis(3-nitro-4-hydroxy-phenylazo)-3-iodobiphenyl and relatednitrophenol derivatives of Chrysamine G are synthesized by substituting3-bromobenzidine (see below) for benzidine and 2-nitrophenol (or othernitrophenol isomers for related derivatives) for methyl salicylate inthe Chrysamine G Synthesis described above. The bromo derivative thusobtained is converted to the tri-alkyl tin and then to the iododerivative as described in detail below.

Alternative Method for the Synthesis of Diazo Compounds

As an alternative method of forming azo compounds, nitroso compounds arecoupled to amines in glacial acetic acid by the Mills reaction. Badger,G. et al., Aust. J. Chem. 17: 1036 (1964). Nitroso compounds are made bythe method of Coleman et al., Organic Synthesis Collective Vol. III:p668. For example, 3-nitrobenzoic acid (Aldrich Chem. Co., Milwaukee,Wis.) (2.44 mmoles) and a solution of 150 mg of NH₄Cl in 5 ml of waterare combined in a 50 ml round-bottomed flask and stirred vigorously.Zinc dust (372 mg) is added in small portions over 5 minutes. After theaddition of zinc is complete the temperature begins to rise, but is keptbetween 50 and 55° C. by the use of an ice bath. The mixture is stirredat that temperature for 20 minutes and then the zinc residues arefiltered and washed with boiling water. The combined filtrate andwashings are cooled to 0° C. in a beaker by the addition of ice. To thiscold mixture of the hydroxylamine, a cold solution of sulfuric acid (750μl of concentrated acid plus enough ice to bring the temperature to −5°C.) is added with stirring. An ice-cold solution of 170 mg of sodiumdichromate dihydrate in 750 μl of water is added all at once withstirring. The resulting 3-nitrosobenzoic acid thus obtained is combinedwith one-half of an equivalent of benzidine, 2,7-diaminofluorene oranother benzidine derivative in glacial acetic acid and warmed to 70-80°C. for 7 hrs and then allowed to stand at room temperature overnight.The mixture is diluted with water and extracted with ethyl acetategiving 4,4′-bis(3-carboxy-phenylazo)-biphenyl),2,7-bis(3-carboxy-phenylazo)-fluorene, or the correspondingbis(3-carboxy-phenylazo) derivative of the particular benzidinederivative employed.

Synthesis of Methoxy Derivatives

Methoxy derivatives of all phenol compounds are synthesized by thefollowing procedure. To one equivalent of the phenolic Chrysamine Gester derivative dissolved in acetone at an approximate concentration of5 mg/ml, is added 10 equivalents of methyl iodide (Aldrich ChemicalCompany, Milwaukee, Wis.) and 10 equivalents of K₂CO₃. The suspension isrefluxed for 6-24 hrs, taken to dryness on a rotary evaporator, andextracted with chloroform. The ester is hydrolysed by addition of 1:1THF/0.1 N NaOH to a concentration of approximately 1 mg/ml and stirringat room temperature from 24-72 hours. Unhydrolysed methoxy-ester isremoved by extraction with chloroform, the pH was adjusted to −2, andthe methoxy-acid was extracted into ethyl acetate.

Synthesis of Substituted Benzidine Derivatives

For the substituted benzidine compounds, the above Chrysamine Gsynthesis general procedure is followed except 3,3′-dichlorobenzidine(Pfaltz & Bauer, Waterbury, Conn.), 4,4′-diaminooctafluorobiphenyl,3,3′,5,5′-tetramethylbenzidine (Aldrich Chemical Company, Milwaukee,Wis.), 3,3′-dimethylbenzidine (Aldrich Chemical Company, Milwaukee,Wis.), 3,3′-dimethoxybenzidine (Aldrich Chemical Company, Milwaukee,Wis.), benzidine-3,3′-dicarboxylic acid (Pfaltz & Bauer, Waterbury,Conn.) or lower alkyl esters is thereof, or 3,3′-dinitrobenzidine (FlukaChemical Corp., Ronkonkoma, N.Y.) are used in place of unsubstitutedbenzidine. Other substituted benzidines including, but not limited to,those listed below are synthesized by the referenced methods and alsocan be used in place of unsubstituted benzidine: 3,3′-dibromobenzidineand 3,3′-diiodobenzidine (Snyder, H. et al., J. Am. Chem. Soc. 71:289-291, 1949); 3-bromobenzidine and 3-iodobenzidine (Badger, G. et al.,Aust. J. Chem. 17: 1036-1049, 1964; Holland, V. et al., Tetrahedron 30:3299-3302, 1974).

Synthesis of Phenanthracene, Benzo(c)cinnoline, Fluorene, Fluorenone,Carbazole, Dibenzofuran, Dibenzothiophene andDibenzothiophene-9,9-dioxide Derivatives

Fluorene (2,2′-methylenebiphenyl) derivatives of Chrysamine G are madeby substituting 2,7-diamino-fluorene (Aldrich Chemical Company,Milwaukee, Wis.) for benzidine in the tetra-azotozation procedures andthen coupled by the same procedures used for tetra-azotized benzidine.Likewise, 2,7-diaminophenanthracene,2,7-diamino-3,6-dimethylbenzo[c]cinnoline, 2,7-diaminofluorenone,9-methyl-2,7-diaminocarbazole, 3,7-diaminodibenzofuran (unlike theremainder of these polycyclic compounds, the conventional numberingsystem puts the oxygen bridge atom of dibenzofurans at the 5-positioninstead of the 9-position, therefore the amino substituents are in the3,7-positions rather than the 2,7-positions as in the other compoundseven though, spatially, the positions are equivalent),2,7-diaminodibenzothiophene, and 2,7-diaminodibenzothiophene-9,9-dioxide(“benzidine sulphone”) are substituted for benzidine in the standardtetra-azotization and coupling procedures. 9-Methyl-2,7-diaminocarbazolecompounds may be N-demethylated after coupling if the unsubstitutedcarbazole is desired. 2,7-Diaminophenanthracene is synthesized byreduction of 2,7-dinitrophenanthracene made from2-amino-3′,5-dinitro-cis-stilbene by the method of Ullmann and MalletBer. 31: 1694-1696, 1898 and Nunn, A. et al., J. Chem. Soc. 1952:2797-2803. 2,7-Diamino-3,6-dimethylbenzo[c]cinnoline is prepared byNaOBr oxidation of the corresponding hydrazo compound formed via thereduction of 3,3′-dimethyl-6,6′-dinitrobenzidine (Aldrich Chemical Co.,Milwaukee, Wis.) with zinc in aqueous NaOH by the method of Snyder etal., J. Amer. Chem. Soc. 71: 289 (1949). Alternatively,3,3′-dimethyl-6,6′-dinitrobenzidine can be first tetra-azotized andcoupled in place of benzidine, followed by treatment with Zn/NaOH andNaOBr as above. 2,7-Diaminofluorenone is synthesized by reduction of2,7-dinitrofluorenone made from 2-amino-3′,5-dinitrobenzophenone by themethod of Ullmann and Mallet Ber. 31: 1694-1696, 1898 and Nunn, A. etal., J. Chem. Soc. 1952: 2797-2803. 9-Methyl-2,7-diaminocarbazole issynthesized by reduction of 9-methyl-2,7-dinitrocarbazole made byN-methylation of 2,7-dinitrocarbazole prepared from2-amino-3′,5-dinitrodiphenylamine as described in Saunders, K. H. andAllen, R. L. M. AROMATIC DIAZO COMPOUNDS 625-640 (Edward Arnold, London,1985), the entire contents of which are hereby incorporated byreference. 3,7-Diaminodibenzofuran is synthesized by reduction of3,7-dinitrodibenzofuran made from 2-amino-3′,5-dinitrodiphenylether asdescribed in Saunders, K. H. and Allen, R. L. M. AROMATIC DIAZOCOMPOUNDS 625-640 (Edward Arnold, London, 1985), the entire contents ofwhich are hereby incorporated by reference. 2,7-Diaminodibenzothiophene,and 2,7-diaminodibenzothiophene-9,9-dioxide are prepared by the methodsof Courtot and Evain Bull. Soc. Chim. 49(iv): 528, 1931 and Cullinaneand Davies Rec. Trav. Chim. 55: 881-886 (1936).

Synthesis of Alkynyl (C≡C) Derivatives of Chrysamine G

5-Iodosalicylic acid (Aldrich Chemical Company, Milwaukee, Wis.) isconverted to the methyl ester by reaction with methanol, trimethylorthoformate and sulfuric acid. The 5-iodosalicylic acid methyl esterthus obtained is reacted with (trimethylsilyl)acetylene (AldrichChemical Company, Milwaukee, Wis.) in the presence of palladium. Thetrimethylsilyl group is removed and two equivalents of the resultant5-acetylenylsalicylic acid methyl ester is reacted with4,4′-dibromobiphenyl (Aldrich Chemical Company, Milwaukee, Wis.) in thepresence of palladium as above. The resultant alkynyl analogue ofChrysamine G,4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylacetylenyl)-biphenyl isprepared by hydrolysis of the ester as described above. This alkynylanalogue is reduced by conventional methods to form the vinyl analogueof Chrysamine G.

Synthesis of Diiodosalicylic Acid Derivative of Chrysamine G

The 3-iodosalicylic acid derivative of Chrysamine G,4,4′-bis(3-carboxy-4-hydroxy-5-iodophenylazo)-biphenyl), is synthesizedby iodination of 5-iodosalicylic acid (Aldrich Chemical Company,Milwaukee, Wis.) at the 3-position followed by selective de-iodinationof the 5-position. After formation of the methyl ester andrecrystallization, the light brown, waxy 3-iodo derivative issubstituted for methyl salicylate in the above procedure for thesynthesis of Chrysamine G. The desired product is separated by elutionfrom a silica gel column with 75% ChCl₃/25% hexane. The ester is thenhydrolysed by dissolution in 1:1 ethanol:H₂O containing four equivalentsof NaOH and refluxed for three hours.

Synthesis of Di-fluoro Chrysamine G

The 5-fluoro derivative,4,4′-bis(2-hydroxy-3-carboxy-5-fluorophenylazo)-biphenyl), issynthesized by substituting 5-fluorosalicylic acid (Aldrich ChemicalCompany, Milwaukee, Wis.) for salicylic acid. [¹⁸F]aryl fluoridesderivatives of Chrysamine G can be prepared by substituting ¹⁸F-labeledprecursors such as [¹⁸F]LiBF₄, in the Schiemann reaction, via triazenedecomposition with Cs [¹⁸F], or via nucleophilic ¹⁸F-for-X substitution,where X=tosyl, triflate, NO₂, ⁺N(CH₃)₃, or halogen. See Fowler, J. andWolf, A. in POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps,M., Mazziota, J., and Schelbert, H. eds.) 391-450 (Raven Press, NY,1986) and Kilbourn, M. Fluorine-18 labeling of radiopharmaceuticals.(Natl. Acad. Press, Washington, D.C.) (1990).

Synthesis of Aromatic Fluoroalkyl and Fluoroalkoxy Derivatives

Aromatic fluoroalkyl derivatives are synthesized employing the method ofBishop et al., J. Med. Chem. 34: 1612 (1991) in which Claisenrearrangement of the appropriate O-allyl ethers forms an aromatic allylderivative which can be further functionalized to yield the fluoroethylor fluoropropyl derivatives. Alternatively, an aromatic iodide can bereadily converted to an aromatic alkyne consisting of two to five carbonatoms in length using the palladium-assisted coupling methodology ofSonogashira et al., Tetrahedron Letters 4467-4470 (1975). Subsequentderivatization of the alkyne yields the fluoroalkyl derivative.Fluoroalkoxy derivatives may be prepared by the method of Chumpradit etal., J. Med. Chem. 36: 21 (1993) in which alkylation of the appropriatephenol with the appropriate 1-bromo (or iodo orsulfonyloxy)-omega-fluoroalkane yields the corresponding fluoroalkoxyderivative.

Radiofluorination of Aromatic Alkylsulfonyloxy and AlkoxysulfonyloxyDerivatives

Radiofluorination to yield the aromatic [¹⁸F]fluoroalkyl and[¹⁸F]fluoroalkoxy derivatives is performed by the method of Mathis etal., Nucl. Med. Biol. 19: 571 (1992) in which aromatic alkyl- oralkoxysulfonyloxy (e.g. alkoxytosylate) derivatives are substituted with[¹⁸F]fluoride to yield aromatic [¹⁸F]fluoralkyl and [¹⁸F]fluoralkoxycompounds.

Radio-Iodination and Radio-Bromination of Chrysamine G Derivatives bythe Tri-Alkyl Tin Route Synthesis of Tri-Alkyl Tin Derivatives ofChrysamine G

The general structure of the tri-alkyl tin derivative of Chrysamine G isshown in FIG. 2B. In general, one tri-alkyl tin group will besubstituted at the 3-position on one side of the biphenyl moiety, butother positions, including the salicylic acid or heterocyclic moiety arealso potential targets. These tri-alkyl tin derivatives are stableimmediate precursors for preparation of the radioiodinated andradiobrominated compounds to be used in humans. More specifically, thesetri-alkyl tin derivatives are used to prepare the halogenatedradioactive compounds applicable for use in in vivo imaging of amyloid.

General Procedures for the Synthesis of Tri-Alkyl Tin Derivatives

Tri-alkyl tin derivatives are prepared from the appropriate arylhalides,[(C₆H₅)₃P]₃Pd(0), and hexaalkylditin by previously published proceduresincluding Kosugi, M., et al., Chem. Lett. 1981: 829; Heck, R. Pure andAppl. Chem. 1978: 691; Echavarren, A. and Stille, J. J. Am. Chem. Soc.1987: 5478; Mitchell, T. J. Organometallic Chem. 1986: 1; and Stille, J.Pure and Applied Chem. 1985: 1771. These derivatives also can beobtained by the use of n-BuLi and trialkyl tin chloride by the procedureof Mathis et al., J. Labell. Comp. and Radiopharm. 1994: 905.

Synthesis of the 3-trialkyl Tin Derivative of4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl

3-Bromo or3-iodo-4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl or itsdimethyl ether are prepared by synthesis of 3-bromo- or 3-iodobenzidine(see above), tetra-azotization and coupling to methyl salicylate as forthe synthesis of Chrysamine G, and methylation of the phenol asdescribed above when the methoxy compound is desired. Under an argonatmosphere, 1 mmol of the phenolic ester or the methoxy ester,[(C₆H₅)₃P]₃Pd(0) (0.1 to 0.2 mmol), hexabutylditin or hexamethyl ditin(1.25 mmol), and dioxane (25 ml) is heated at 70° C. for 16 hrs. Thereaction mixture is cooled and the solvent is evaporated. Tri-alkyl tinhalide is removed with aqueous KF. The organics are extracted with ethylacetate, dried over magnesium sulfate, filtered, and the solvent isevaporated under reduced pressure. The residue is purified on silica gelto obtain3-trialkyltin-4,4′-bis(3-methoxycarbonyl-4-hydoxyphenylazo)-biphenyl.

Radio-Iodination or Radio-Bromination of Tri-Alkyl Tin Derivatives

The tributyl or trimethyl tin derivatives are radio-iodinated withNa[¹²⁵I] or Na[¹²³I] or radio-brominated with Na[⁷⁵Br] or Na[⁷⁶Br] bypublished procedures such as Mathis et al., J. Labell. Comp. andRadiopharm. 1994: 905; Chumpradit et al., J. Med. Chem. 34: 877 (1991);Zhuang et al., J. Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med.Chem. 37: 4245 (1994). In general, 0.5 mg of tri-alkyl tin compound, 0.2ml of anhydrous acetonitrile, 10 μl of 2M H₃PO₄, 2-100 μl of a solutionof high specific activity (>2000 Ci/mmol) Na[¹²⁵I] or Na[¹²³I] (or Na[⁷⁵Br] or Na[⁷⁶Br]) in pH 9-12 NaOH, and dichloramine-T (DCT) (20 μl of2.5 mg/ml DCT in acetonitrile) are placed in a 1 ml Reacti-Vial. Thevial is capped and the mixture is stirred at room temperature in thedark. The reaction is monitored by HPLC and after 30 min is quenchedwith 50 μl of 2 M Na₂S₂O₃. The product is purified by standardchromatographic techniques. Mathis et al., J. Labell. Comp. andRadiopharm. 1994: 905. Similarly, low specific activity ¹⁸F derivativesare prepared by analogous procedures.

General Procedures for the Preparation of Non-Radioactive I, Br, Cl, Fand -SH Derivatives

In general, 3- or 4-amino derivatives of salicylic acid, or thecorresponding derivatives of the heterocyclic analogues of salicylicacid shown in FIG. 2, are converted to the corresponding diazo compoundswith sodium nitrite and HCl or H₂SO₄. The iodine derivatives aredirectly prepared by forming the diazonium iodide which is thenconverted into the aryl iodide, or by way of the triazene intermediates.See, e.g., Greenbaum, F. Am. J. Pharm. 108: 17 (1936), Satyamurthy, N.and Barrio, J., J. Org. Chem. 48: 4394 (1983) and Goodman, M. et al., J.Org. Chem. 49: 2322 (1984). Aryl bromides and chlorides are preparedfrom the diazo compounds by treatment with CuCl or CuBr according to theSandmeyer reaction or via the triazene as for the iodine derivatives.Aryl fluorides are prepared by treating the diazonium compounds withNaBF₄, HBF₄, or NH₄BF₄ according to the Schiemann reaction or viatriazene decomposition similar to the iodine derivatives. Aryl thiolsare prepared from the diazonium compounds by treatment withsulfur-containing nucleophiles such as HS⁻, EtO−CSS⁻, and S₂ ²⁻.Alternatively, aryl thiols can be prepared by replacement of arylhalides with sulfur containing nucleophiles. These reactions aredescribed in March, J., ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985).

General Procedures for the Preparation of Radioactive C, F and TcDerivatives

In addition to the above procedures, high specific activityradiolabeling with ^(99m)Tc for SPECT or with the positron-emittingradionuclides ¹¹C, ¹⁸F, ⁷⁵Br and ⁷⁶Br is accomplished according toliterature-based methods well known in the art. Some of the potentialspecific methods are described below, but there are other well-knownmethods which will be apparent to those skilled in the art and aredescribed in Fowler, J. and Wolf, A. Positron emitter-labeled compoundsin POSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M.,Mazziota, J., and Schelbert, H. eds.) p 391-450 (Raven Press, NY)(1986), Coenen, H. et al., Radiochimica Acta 34: 47 (1983), andKulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991), thecontents of which are hereby incorporated by reference.

^(99m)Tc derivatives are prepared by complexation with the aryl thiols.Radiolabeling with ¹¹C can be readily done via N-methylation,O-methylation as described above substituting [¹¹C]methyl iodide,[11C]alkylation, or [¹¹C] carboxylation of suitable Chrysamine Ganalogues. [¹⁸F]aryl fluorides derivatives can be prepared bysubstituting ¹⁸F-labeled precursors such as [¹⁸F]LiB₄ in the Schiemannreaction described above, via triazene decomposition with Cs[¹⁸F], orvia nucleophilic ¹⁸F-for-X substitution, where X=tosyl, triflate, NO₂,⁺N(CH₃)₃, or halogen. Radiobromination using ⁷⁵Br and ⁷⁵Br can beaccomplished using either electrophilic (Br⁺) or nucleophilic (Br⁻)substitution techniques analogue to radioiodiantion techniques, seeCoenen, H., supra.

Synthesis of the 3-Hydroxy-1,2-benzisoxazole Derivative and RelatedDerivatives (see FIG. 2C)

2,6-Dihydroxybenzoic acid (γ-resorcylic acid) methyl ester (TCI America,Portland, Oreg.) is converted to the hydroxamic acid by the use ofhydroxylamine hydrochloride according to the method of Böshagen (Chem.Ber 100: 954-960; 1967). The hydroxamic acid is converted to thecorresponding 3-hydroxy-1,2-benzisoxazole with the use of SOCl₂ and thentriethylamine, also by the method of Böshagen (Chem. Ber 100: 954-960;1967). This compound is then coupled with benzidine, 3-bromobenzidine,or other benzidine derivatives via the same procedure used for salicylicacid derivatives. The bromo derivatives can then converted to thetri-alkyl tin and iodo-derivatives as described above.

Alternatively, methyl salicylate is coupled to benzidine as usual andthe resulting 4,4′-bis(3-methoxycarbonyl-4-hydroxyphenylazo)-biphenyl(or the dimethyl ester of Chrysamine G) is converted to the hydroxamicacid and then the benzisoxazole by the method of Böshagen as describedabove. A third type of 3-hydroxy-1,2-benzisoxazole is synthesized fromthe dimethyl esters of several isomeric dihydroxy benzenedicarboxylicacids including 4,6-dihydroxy-1,3-benzenedicarboxylic acid,3,6-dihydroxyphthalic acid, and 2,5-dihydroxyterephthalic acid (AldrichChem. Co., Milwaukee, Wis.). After coupling to benzidine or itsderivatives by standard procedures, the dihydroxy/diesters is convertedto dihydroxy/dihydroxamic acids by reaction with hydroxylamine by themethod of Böshagen described above. Conversion to the doublebenzisoxazole is effected by treatment with SOCl₂ and triethylamine,again, by the method of Böshagen described above.

Synthesis of the Phthalimide or Isoindole-1,3(2H)-dione Derivative (seeFIG. 2D)

3-Hydroxyphthalimide made from 3-hydroxyphthalic anhydride (AldrichChemical Company, Milwaukee, Wis.) is coupled with benzidine or itsderivative via the same procedure used for salicylic acid derivativesand then converted to the tri-alkyl tin and iodo-derivatives asdescribed above.

Synthesis of the Phthalhydrazide or 2,3-benzodiazine-1,4(2H,3H)-dioneDerivative (see FIG. 2E)

3-Hydroxyphthalhydrazide made from the reaction of 3-hydroxyphthalicanhydride (Aldrich Chemical Company, Milwaukee, Wis.) with hydrazine iscoupled with benzidine or its derivative via the same procedure used forsalicylic acid derivatives and then converted to the tri-alkyl tin andiodo-derivatives as described above.

Synthesis of the 2,3-benzoxazine-1,4(3H)-dione Derivative (see FIG. 2F)

3-Hydroxyphthalic anhydride (Aldrich Chemical Company, Milwaukee, Wis.)is converted to the 2,3-benzoxazine with the use of hydroxylamine. Thebenzoxazine derivative is then coupled with benzidine or its derivativevia the same procedure used for salicylic acid derivatives and thenconverted to the tri-alkyl tin and iodo-derivatives as described above.

Synthesis of the (2H)1,3-benzoxazine-2,4(3H)-dione Derivative (see FIG.2G)

This compound is synthesized by the method of Effenberger et al., (Chem.Ber. 105: 1926-1942; 1972). Briefly, phenol is coupled with benzidine orits derivative via the same procedure used for salicylic acidderivatives. This adduct is then converted to the carbamate by reactionwith ethoxycarbonylisocyanate (O═C═N—CO—O—Et) in the presence oftriethylamine. This substituted carbamate (or N-ethoxycarbonyl-carbamicacid-phenyl ester) is converted to the benzoxazinedione by heating indiphenyl ether. The benzoxazinedione is then converted to the tri-alkyltin and iodo-derivatives, as described above.

Synthesis of the (3H)2-benzazine-1,3(2H)-dione Derivative (see FIG. 2H)

3-Hydroxyphenylacetic acid (Aldrich Chemical Company, Milwaukee, Wis.)is converted to the amide and then coupled with benzidine or itsderivative via the same procedure used for salicylic acid derivatives.This adduct is then converted to the N-(3-hydroxyphenylacetoxy)-carbamicacid ethyl ester derivative by reaction with ethyl chloroformate. Thissubstituted carbamate is converted to the benzazinedione by heating indiphenyl ether. The benzazinedione is then converted to the tri-alkyltin and iodo-derivatives, as described above.

Synthesis of the 1,8-Naphthalimide Derivative (See FIG. 2I)

4-Amino-1,8-naphthalimide is coupled with benzidine or its derivativevia standard procedures for diazo coupling of aryl amines, as describedin Saunders, K. H. and Allen, R. L. M. AROMATIC DIAZO COMPOUNDS (EdwardArnold, London, 1985), the entire contents of which are herebyincorporated by reference. The diazo naphthalimide is then converted tothe tri-alkyl tin and iodo-derivatives, as described above.

Synthesis of Tetrazole and Oxadiazole Derivatives (See FIGS. 2J and 2K)

2-Cyanophenol (Aldrich Chemical Company, Milwaukee, Wis.) is convertedto the tetrazole by reaction with sodium or aluminum azide according tothe method of Holland and Pereira J. Med. Chem. 10: 149 (1967) andHolland U.S. Pat. No. 3,448,107. Briefly, 2-cyanophenol or cyanophenolderivatives of Chrysamine G (1 mmol) in 40 ml DMF is treated with sodiumazide (10 mmol) and triethylamine hydrochloride (10 mmol) under argon.The mixture is stirred at 120° C. for 2 hrs after which the mixture iscooled and worked up in a manner analogous to that described above forChrysamine G.

The oxadiazoles are synthesized by treatment of the tetrazoles preparedas above with an acid anhydride (such as acetic anhydride). An alternatemethod is that of Bamford et al., J. Med. Chem. 38: 3502 (1995). In thisprocedure, hydrazide derivatives of Chrysamine G or salicylic acid(obtained by treatment of the respective esters with hydrazine) aretreated with methyl isothiocyanate in the presence ofdicyclohexylcarbodiimide.

Synthesis of Chrysamine G Derivatives for Use as Controls

The aniline derivative is synthesized by substituting two equivalents ofaniline (Fisher Chemical Co., Fair Lawn, N.J.) for each equivalent ofbenzidine. The 2,2′-disulfonic acid derivative is synthesized bysubstituting benzidine-2,2′-disulfonic acid (Pfaltz & Bauer, Inc.,Waterbury, Conn.) for benzidine. The phenol derivative is synthesized bysubstituting one equivalent of phenol for each equivalent of salicylicacid. Congo red (Aldrich certified grade) is obtained commercially.

EXAMPLE 2 Chrysamine G and Chrysamine G Derivatives Bind Specifically toAβ Binding to Synthetic Aβ (10-43)

Chrysamine G binds well to synthetic Aβ (10-43) peptide in vitro. FIG.4A shows a Scatchard analysis of the binding of Chrysamine G to Aβ(10-43). The higher affinity component has a K_(D) of 0.257 μM and aB_(max) of 3.18 nmoles Chrysamine G/mg Aβ (10-43). The lower affinitycomponent is less well defined by these data, but appears to have aK_(D) of 4.01 μM and a B_(max) of 18.7 nmoles Chrysamine G/mg Aβ(10-43). The low affinity component represents the binding of ChrysamineG at high concentrations to a distinct, low-affinity site, not thebinding to an impurity in the preparation. The amount of Chrysamine Ginjected in vivo is so low that there is not any binding to thelow-affinity component. At very low concentrations, the ratio ofhigh-to-low affinity binding is very large.

The amount of Chrysamine G binding is linear with peptide concentrationover the range employed, as shown in FIG. 5.

Kinetics of Binding

Kinetic studies showed a fairly rapid association (FIG. 6A), essentiallycomplete by 1 min, at a [Chrysamine G]=112 μM with a t_(½) of 8.9±1.8sec and a somewhat less rapid dissociation (FIG. 6C), t_(½)=55±9.4 sec[dissociation rate constant (k⁻¹)=1.26×10⁻² sec⁻¹]. FIG. 6B shows atransformation of the association kinetic data according to the methodof Bennett and Yamamura. Bennett, J. P. and Yamamura, H. I. inNEUROTRANSMITTER RECEPTOR BINDING (N.Y.: Raven Press 1985) pp. 61-89.The linear portion of the association curve in FIG. 6A is transformedinto the line of FIG. 6B, in which ln[B_(eq)/(B_(eq)−B_(t))] is plottedversus time, where B_(eq) is the amount of Chrysamine G bound atequilibrium (4 min) and B_(t) is the amount bound at time=t. The slopeof this line equals k_(observed) and k₁=(k_(observed)−k⁻¹)/[ChrysamineG], where k⁻¹ is the dissociation rate constant determined from the datain FIG. 6C. The curve in FIG. 6C follows the equation:

A _(t) =A ₀ e ^(−k) ^(⁻¹) ^(t)

where A_(t) is the amount of Chrysamine G remaining bound at time=t, A₀is the amount of Chrysamine G bound at time=0, t is the time in min, andk⁻¹ is the dissociation rate constant. From this analysis, theassociation rate constant (k₁) is calculated to be 3.75×10⁴ M⁻¹ sec⁻¹giving a K_(D)=k⁻¹/k₁=0.34 μM, in good agreement with the Scatchardanalysis.

Chrysamine G Derivatives Can Inhibit the Binding of Chrysamine G to Aβ

K_(i) values for the inhibition of [¹⁴C]Chrysamine G binding to Aβ(10-43) by the Chrysamine G analogues are shown under the chemicalstructures in FIG. 1 and several displacement curves are shown in FIG.3. K_(i) is defined as IC₅₀/(1+[L]/K_(D) ), where [L] is theconcentration of [¹⁴C] Chrysamine G in the assay (0.0100-0.125 μM) andK_(D) is 0.26 μM, the K_(D) of Chrysamine G determined by the Scatchardanalysis above. Chrysamine G itself gives a K_(i) of 0.37±0.04 μM, avalue very consistent with those obtained from the Scatchard and kineticanalyses. Congo red gives a K_(i) of 2.82±0.84 μM. The difluoroderivative of Chrysamine G, (5-FSA)CG, (FIG. 1) is one-third as potentas Chrysamine G itself (K_(i)=1.16±0.19 μM). The activity of thedifluoro Chrysamine G derivative suggests that an ¹⁸F difluoroChrysamine G derivative works for PET imaging and an ¹⁹F difluoroChrysamine G derivative works for MRS/MRI imaging of brain.

The 3-ICG is slightly more potent than Chrysamine G. The activity of the3-ICG derivative suggests that an ¹²³I difluoro Chrysamine G derivativeworks for SPECT imaging. Methylating the phenol of 3-ICG decreases theaffinity by a factor of 10 in 3-IGC(OMe)₂. Methylating the carboxylategroup effected an even greater (about 200-fold) decrease in affinity inCG(COOMe)₂. Removing the acid moiety entirely, as in the phenolderivative, completely destroyed binding affinity.

These results suggest that the acid moiety of Chrysamine G analoguesplays the major role in binding to Aβ and that the phenol moiety plays afacilitating role. The effect of the phenol could occur through hydrogenbonding to the acid which could serve to stabilize the structuralorientation of the acid moiety. The presence of a phenol in the orthoposition could also alter the charge distribution of the acid eitherthrough hydrogen bonding or through changes in the charge distributionof the aromatic system as a whole. Alternatively, the phenol coulddirectly participate in binding to the amyloid via a bi-dentateattachment of both the phenol and the acid to the amyloid binding site.Adding a second phenol ortho to the carboxylate as in the resorcylicacid derivative, (6-OHSA)CG, produces the highest affinity compound inthis series having a K_(i) of 0.094±0.02 μM.

Increasing the lipophilicity of the biphenyl backbone appears toincrease the affinity somewhat. The di-halo derivatives, 3,3′-I₂CG,3,3′-Br₂CG, and 3,3′-Cl₂CG, all have very similar K_(i) values which areabout half that of Chrysamine G.

Distorting the dihedral angle between the phenyl rings of the biphenylgroup by substitution at the 2-position markedly diminishes affinity.This is demonstrated by the inactivity of the 2,2′-di-sulfonic acidderivative of Chrysamine G, 2,2′-(SO₃)₂CG. Since the 3,3′di-carboxylicderivative, 3,3′-(COOH)₂CG, shows only a 7-fold loss of activity fromChrysamine G, it is unlikely that the additional acidic moieties are thesole cause for the loss of activity in the 2,2′-disulfonic acid. This2,2′-derivative is unique in that the bulky sulfonate groups in the2-position force the biphenyl group out of planarity. Molecularmodelling studies showed that the dihedral angle between the twobiphenyl benzene rings in the 2,2′-disulfonic acid derivative is 83°.This angle is approximately 35-40° in Chrysamine G and all of the otheractive derivatives.

In an attempt to explore the importance of the bidentate nature of thefunctional groups of Chrysamine G, the binding of an aniline derivativewhich represents one-half of a Chrysamine G molecule (FIG. 1) wasstudied. An approximation of the energy of binding can be calculatedfrom the equation:

ΔG=−RT ln K _(eq)

where ΔG is the energy of the binding reaction, R is the molar gasconstant [8.31441 J/(mole·°K)], T is temperature in °K and K_(eq) is theequilibrium constant for the reaction:

[probe]+[peptide]⇄[probe·peptide]

and K_(eq)=1/K_(D)≈1/K_(i). Using the value of 0.26 μM for the K_(D) ofChrysamine G, the energy of binding is roughly 38 KJ/mole. If theaniline derivative binds with one-half of this energy, the expectedenergy of binding would be about 19 KJ/mole. From the K_(i) of 73 μM forthe aniline derivative, the energy of binding is 23 KJ/mole which is inacceptable agreement with the predicted value. The importance of thehydrophobic region of Chrysamine G and the aniline derivative isdemonstrated by the total lack of binding activity of salicylic aciditself.

The affinity of Chrysamine G for Aβ appears to be several fold greaterthan the affinity of Congo red for this peptide. The binding isreversible with a dissociation constant of approximately 250-400 nM,whether measured by Scatchard analysis, kinetic methods, or inhibitionof binding. Owing to the non-crystalline, poorly soluble nature ofamyloid fibrils, the structure of Congo red or Chrysamine G complexeswith amyloid has never been defined by precise structural techniquessuch as x-ray crystallography or multi-dimensional NMR. Models of Congored interactions with amyloid have been proposed. Cooper, Lab. Invest.31: 232 (1974); Romhanyi, Virchows Arch. 354: 209 (1971). This worksuggests that Congo red does not bind to a single amyloid peptidemolecule, but binds across several Aβ molecules oriented by virtue ofthe beta-sheet fibril. Klunk et al., J. Histochem. Cytochem. 37: 1273(1989).

FIG. 7 shows a schematic of this model, generated using MacroModel 2.5,in which Chrysamine G spans 5 peptide chains in an anti-parallelbeta-sheet conformation. The peptides are used without furtherstructural refinement. The peptides are aligned so that alternate chainswere spaced 4.76 Å apart, characteristic of beta-sheet fibrils.Alternate peptide chains are drawn in black and white. Chrysamine G(black) is energy minimized and aligned with the fibril model tomaximize contact with lysine-16 (light grey ovals in top figure) and thehydrophobic phenylalanine 19/20 region (bottom). The two views are ofthe same model at approximately 90° from one another. The white arrowsindicate the direction taken to obtain the alternate view.

The 19.1 Å spacing between the carboxylic acid moieties of Chrysamine Gmatches well with the distance of 19.0 Å across the 5 chains (4×4.76 Åbetween adjacent chains shown by Kirschner et al., Proc. Natl. Acad.Sci. U.S.A. 83: 503 (1986)). If the native structure of Aβ involves ahairpin loop structure as Hilbich et al., suggest (Hilbich et al., J.Mol. Biol. 218: 149 (1991)), then chains 1 and 2, 3 and 4, 5 and 6,etc., would be folded halves of the same molecule, but the model wouldotherwise be the same. Also important to note is the necessity forpositively charged amino acid residues in this model, such as lysine-16in Aβ. Previous work has shown that Congo red binding correlates wellwith the number of positively charged amino acids in a sample of amyloidfibrils. Klunk et al., J. Histochem. Cytochem. 37: 1273 (1989). Thebidentate nature of the model in FIG. 7 and the importance ofhydrophobic interactions is supported by the decrease in affinity of themono-dentate aniline analogue of Chrysamine G and the inactivity ofsalicylic acid as well as the increased potency of the more lipophiliccompounds having two halogens on the benzidine moiety (see FIG. 1). Theimportance of the nearly planar biphenyl group is suggested by theinactivity of the 2,2′-disulfonic acid derivative.

EXAMPLE 3 Chrysamine G Distinguishes Alzheimer's Disease Brain fromNormal Brain Characterization of the Binding of Chrysamine G to AD Brain

Scatchard analyses of the binding of Chrysamine G and Chrysamine Gderivatives to AD brain samples were performed in an effort tounderstand the increased binding of Chrysamine G to AD brain. (FIG. 4B,Table 1). Under the conditions employed, control and AD brain showed asingle binding component. The K_(D) in AD brain was 16% lower thancontrol but the difference was not significant (p=0.29). The B_(max) inAD brain was 36% higher than the B_(max) in control brain, but, again,the difference did not reach significance (p=0.09). Therefore, theincreased binding in AD brain appears to be mainly due to the presenceof more of the same binding component which exists in control brain,rather than the presence of a unique component.

TABLE 1 Comparison of binding Parameters in AD and control brain B_(max)(pmol/μg K_(D) (μM) prot) Control 0.47 ± .049 0.576 ± .092 (n = 6) AD (n= 5) 0.39 ± .048 0.784 ± .061

The binding of CG to AD brain significantly correlated with numbers ofNPs in the association cortices of the brain. FIG. 8A shows thecorrelation of [¹⁴C]CG binding with numbers of NPs in thesuperior/middle frontal and superior temporal cortex of AD brain. Thecorrelation with NPs was significant whether controls were included(r=0.69; p=0.001) or if the AD brains were considered alone (r=0.59;p=0.007). FIG. 8B shows a similar correlation with NFT counts. As withNPs, the correlation with NFTs was significant whether controls wereincluded (r=0.60; p=0.001) or if the AD brains were considered alone(r=0.50; p=0.026). The correlation with NFTs is not surprising since CGis a derivative of Congo red, which stains NFTs. The number of NPs wassignificantly correlated with the number of NFTs (r=0.82; p=0.0001).

Only qualitative data on the presence or absence of amyloid angiopathywas available for the brains used in this study, so similar correlationscould not be performed between CG binding and cerebrovascular amyloidlevels. The presence of amyloid angiopathy does appear to be aconfounding variable in the correlation of CG binding with NP counts.FIG. 8A shows the improved correlation of CG binding to NP counts inbrains without amyloid angiopathy (r=0.79; p=0.01) compared to thosebrains with cerebrovascular amyloid deposits (r=0.49; p=0.15). A similarimprovement was not found in the correlation to NFT counts.

The K_(D) for [¹⁴C]Chrysamine G binding to AD brain is similar to thatfound for [¹⁴C]Chrysamine G binding to synthetic Aβ in vitro, suggestingthat binding in brain homogenates also may represent interaction withAβ. The correlation of Chrysamine G binding to NFTs may indicate thatChrysamine G binds to these structures in brain homogenates as well.Alternatively, since the number of NFTs correlates closely with thenumber of NPs, the correlation of [¹⁴C]Chrysamine G binding to NFTs mayjust be an epiphenomenon of Chrysamine G binding to NPs.

The useful Chrysamine G derivatives or analogues provided herein havebinding affinities that are at least in the range of 0.01 to 10.0 μMK_(D), as measured by binding to either synthetic Aβ peptide orAlzheimer's Disease brain tissue; higher affinity compounds havingbinding affinities in the range of 0.0001 to 0.01 μM are also useful inthe method of the present invention.

Considering the above, Chrysamine G binding may not be specific for Aβ.Instead, Chrysamine G binding may reflect the total amyloid “load” ofthe brain, comprised of aggregated deposits of Aβ in neuritic plaquesand cerebrovascular amyloid. Deposits of phosphorylated tau protein inNFTs may contribute to Chrysamine G binding as well. Goedert, M. et al.,PNAS 85: 4051 (1988). NFTs also are composed of anti-parallel beta-sheetfibrils similar in quaternary structure to fibrils of Aβ. Kirschner etal., Proc. Natl. Acad. Sci. U.S.A. 83: 503 (1986).

Total and Relative Chrysamine G Binding Distinguishes AD From NormalBrain

In vitro binding assays such as those described above and below arewidely used in the art as models to screen compounds for in vivo bindingin brain and to predict success in subsequent in vivo imaging studies.See, Young, A. et al., Receptor Assays: In Vitro and In Vivo. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) pp. 73-111 (1986). The labeled Chrysamine Gand Chrysamine G derivatives of the invention also may be used in the invitro binding assays described above and below to quantitate amyloiddeposits in biopsy or post-mortem specimens.

Saturable (specific) binding of [¹⁴C] Chrysamine G was observed both inAD brain and control brain homogenates and constituted 60-80% of totalbinding in AD brain. Non-saturable binding was very similar in AD andcontrol brain. Both saturable and total binding were greater in AD brainthan in control. Despite the lower sensitivity obtained when using totalbinding, this parameter is more predictive of success in in vivo studieswhich are the ultimate goal of this invention. Also for the purpose ofextension to in vivo studies, it is advantageous if Chrysamine G bindingin cortical areas is normalized to a brain area in which Chrysamine Gbinding is very similar in both AD and control brain. This obviates theneed to calculate the absolute quantity bound which is difficult to doin vivo. We examined binding in the cerebellum as a potential controlarea because classical NPs are exceedingly rare in this brain area(Joachim et al., Am. J. Pathol. 135: 309 (1989)).

The average amount of [¹⁴C]Chrysamine G bound to control cerebellum isnearly identical to the amount bound to AD cerebellum (Table 2),supporting the use of cerebellum as an internal control. Therefore, thecerebellar ratio (CBR) accurately reflects the absolute quantity of[¹⁴C]Chrysamine G bound and offers the advantage of providing aninternal control for each brain. Binding is greater in AD brain whetherexpressed in absolute terms of fmol/μg protein (Table 2) or as a ratioto the binding in the cerebellum of the same brain (Table 3). The CBR isthe more sensitive measure and shows less variability between brains.The use of total binding and CBRs greatly facilitates extension of theseex vivo results to in vivo studies. Accordingly, the results below areexpressed in these terms whenever appropriate.

TABLE 2 Comparison of total binding in AD and control brain*. Control ADBrain Area (fmol/μg protein) (fmol/μg protein) p Value Cerebellum 75 ±13 (n = 8)  73 ± 9 (n = 11) p = 0.91 Frontal Pole 58 ± 8 (n = 6) 124 ±16 (n = 10) p < 0.006 Superior/Middle 54 ± 10 (n = 8) 130 ± 21 (n = 11)p < 0.005 Frontal Superior 66 ± 17 (n = 8) 121 ± 14 (n = 11) p < 0.02Temporal Head of Caudate 73 ± 11 (n = 4) 123 ± 22 (n = 7) p = 0.14Inferior 76 ± 13 (n = 8) 137 ± 19 (n = 11) p < 0.03 Parietal Occipital64 ± 16 (n = 8)  95 ± 12 (n = 11) p = 0.15 *High and low-plaque ADbrains combined.

TABLE 3 Comparison of total binding in AD and control brain as a ratioto cerebellum*. Control AD Brain Area (CBR) (CBR) p Value Frontal Pole0.87 ± .04 (n = 6) 1.87 ± .25 (n = 10) p < 0.004 Superior/Middle 0.73 ±.02 (n = 8) 1.84 ± .18 (n = 11) p < 0.001 Frontal Superior 0.86 ± .08 (n= 8) 1.63 ± .17 (n = 11) p < 0.002 Temporal Head of Caudate 0.95 ± .04(n = 4) 1.76 ± .31 (n = 7) p < 0.04 Inferior 0.90 ± .08 (n = 8) 1.93 ±.20 (n = 11) p < 0.001 Parietal Occipital 0.77 ± .13 (n = 8) 1.44 ± .20(n = 11) p < 0.02 *The CBR for each sample is obtained by dividing theabsolute value of [¹⁴C] Chrysamine G binding in that sample by theabsolute value of [¹⁴C] Chrysamine G binding in the cerebellar samplefrom that same brain. The values in the table are the average CBRs fromeach brain area (±SEM). High- and low-plaque AD brains combined.

FIG. 9A and 9B shows the binding of [¹⁴C]Chrysamine G to six brain areasnormalized to the cerebellum of the same brain. The binding ofChrysamine G to AD brain areas in AD brains having more than 20 NPs/×200magnification, “High Plaque AD Brains”, is shown in FIG. 9A. The bindingof Chrysamine G to AD brain areas in AD brains having less than 20NPs/×200 magnification, “Low Plaque AD Brains”, is shown in FIG. 9B. Inall brain areas, the binding to AD brain is significantly greater thanthe binding to control (see Table 3). In superior/middle frontal cortex,there is no overlap between control and any of the AD samples. In allbrain areas except the occipital cortex, there is no overlap betweencontrol and the AD samples having >20 NPs/×200 magnification. In brainareas with the least deposition of classical NPs, such as the occipitalcortex (and cerebellum), the greatest overlap between AD and control wasobserved.

FIG. 9C shows the data from two patients who had Down's syndrome. Down'ssyndrome patients all develop deposits of Aβ by their fourth decade andmany develop AD. Wisniewski et al., Neurology 35: 957 (1985); Schapiroet al., Neurobiol. Aging 13, 723 (1992). Both of these patients showed[¹⁴C]Chrysamine G binding above the control range. Since the youngerpatient (23 years old) had amyloid deposits but was not yet clinicallydemented, FIG. 9C suggests that Chrysamine G can detect differences fromcontrol in non-demented patients destined to develop AD long before thedementia is clinically evident.

The compounds and method of the invention provide two usefulmeasurements for differentiating AD brain from normal brain; either (1)total Chrysamine G binding (Table 2) or (2) the ratio of Chrysamine Gbinding in a given brain area to binding in the cerebellum of the samebrain (Table 3). These measurements furnish two great advantages for invivo quantitation of AD neuritic plaques. First, by providing a means tomeasure total Aβ binding, rather than specific Aβ binding, the instantinvention can quantify Aβ deposition without having to expose thesubject to a second injection of radioactive material in order tomeasure non-specific binding. Because of this, the data are expressed astotal binding only. In all of the experiments presented, specificbinding data yields even greater differences between AD and controlbrain.

Second, variations in brain uptake of Chrysamine G derivatives willaffect the absolute concentration of Chrysamine G in brain. Somemechanism will be necessary, therefore, to account for these variationsbetween subjects. Each patient can serve as his/her own control byfinding a brain area that shows little Aβ deposition (i.e., anexperimental “blank”). Since classical NPs are exceedingly rare in thecerebellum (Joachim et al., Am. J. Pathol. 135: 309 (1989)), ChrysamineG binding to the cerebellum was used as a control for each brainstudied. The results were expressed in terms of the ratio of ChrysamineG binding in a given brain area to binding in the cerebellum of the samebrain (FIG. 9 and Table 3).

For the purposes of in vivo quantitation of amyloid in AD, the effect ofbrain atrophy should be considered. Therefore, when using the ChrysamineG and Chrysamine G derivative probes in vivo to quantitate amyloid,brain atrophy can be corrected based on MRI volume measurements. MRIvolume measurements performed in conjunction with the method of theinvention are analogous to those routinely employed in the art. See,Pearlson, G. and Marsh, L. MAGNETIC RESONANCE IMAGING IN PSYCHIATRY inAnnual Review of Psychiatry (Vol. 12) Oldham, J. et al., eds. p. 347-381(1993). Therefore a method for determining the total radioactivity pervolume of brain area would use the following equation:

total SPECT or PET signal from brain area “A” MRI determined brainvolume (excluding CSF) in brain area “A”

Designating this measurement as the signal/volume for brain area “A” orS/V_(A) means that the cerebellar ratio would be expressed as:${Ratio}_{A} = \frac{S/V_{A}}{S/V_{CB}}$

where S/V_(CB) is the signal/volume in the cerebellum of the samesubject during the same imaging study. This ratio from any brain areaother than cerebellum from a patient suspected of having AD or otherpathological condition characterized by the deposition of amyloid couldthen be compared to the normal range of the analogous ratio from thesame brain area of a group of age-matched normal control subjects. Theratio of the binding to brain areas with high deposits of neuriticplaques to the cerebellum can be used as the parameter to distinguishAlzheimer from control subjects.

EXAMPLE 4 The Octanol-Water Partition Coefficients of Chrysamine G,Chrysamine G Derivatives, and Congo Red

The octanol-water partition coefficient is a measure of the relativelipophilicity of a compound. The more lipophilic a compound, the morelikely it is to cross the blood-brain barrier. See, Goodman and Gilman'sTHE PHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.). The octanol/waterpartition coefficient of Chrysamine G is 60.22±3.97 and that of Congored is 0.665±0.037 (p<0.001). This suggests that Chrysamine G isapproximately 90 times more lipophilic than Congo red and therefore istheoretically more likely to cross the mammalian blood-brain barrier.The octanol/water partition coefficients for the 3-iodo and 3,3′-diiododerivatives of Chrysamine G (FIG. 1) are 72.53±0.74 and 112.9±7.3,respectively. These octanol/water partition coefficients show that thesederivatives, which are non-radioactive analogues of some of theradiolabeled Chrysamine G derivatives to be used for in vivo studies,are up to 170 times more lipophilic than Congo red and up to twice aslipophilic as Chrysamine G. This suggests they will enter the brain muchbetter than either Congo red or Chrysamine G.

EXAMPLE 5 The Ability of Chrysamine G and Chrysamine G Derivatives toCross the Blood-Brain Barrier and Metabolism of Chrysamine G

Use of the amyloid probes to diagnose AD in vivo requires them to beable to cross the blood-brain barrier and gain access to parenchymalamyloid deposits.

The ability of Chrysamine G to cross the blood-brain barrier was studiedin Swiss-Webster mice. After i.v. injection, the brain/blood ratiomeasured at 15 min was over 10:1 and approached 20:1 by 35 min (FIG.10). The radioactivity in brain stayed nearly constant over this period,but decreased in the blood and increased in the liver. The brain/kidneyratio was highest at 15 min (over the time points sampled) andapproached 0.5. When brain and liver were extracted 60 min after i.v.injection of [¹⁴C]Chrysamine G, >95% of the recovered radioactivityco-eluted with authentic Chrysamine G on reverse phase HPLC, indicatingno significant metabolism of Chrysamine G over this period of time.

Chrysamine G does get into normal mouse brain, and the brain/blood ratiois high. The radioactivity in brain remained relatively constant overthe first 30 min while decreasing in blood and increasing in liver. Thissuggests that the high brain/blood ratio is more a result of efficientremoval of Chrysamine G from the blood by the liver than to furtheraccumulation in the brain. At 60 min, essentially all of theradioactivity found in the brain and liver proved to be unchangedChrysamine G. Congo red does not cross the blood-brain barrier well.Tubis et al., J. Amer. Pharm. Assn. 49: 422 (1960). Most of the Congored is cleared by the liver and spleen and the brain/kidney ratioachieved in guinea pigs is approximately 0.07. Tubis et al., supra.Chrysamine G also is cleared by the liver, but has greater entry intothe brain.

In vivo animal testing provides yet a further basis for determiningdosage ranges, efficacy of transfer through the blood barrier andbinding ability. Particularly preferred for this purpose are thetransgenic mouse model of Games et al., (Nature 373: 523 (1995)) and the“senile animal” model for cerebral amyloidosis; i.e., animals such asthe transgenic mice or aged dogs or monkeys, which are known to developvariable numbers of Alzheimer-type cerebral neuritic plaques, seeWisniewski et al., J. Neuropathol. & Exp. Neurol. 32: 566 (1973), Selkoeet al., Science 235: 873 (1987), are tested for binding and detectionefficacy. This in vivo assay requires control-biopsy or necropsymonitoring to confirm and quantify the presence of amyloid deposits.

Other suitable animal models for use in testing the compositions andmethods of the present invention are produced transgenically. Forinstance, Quon et al., Nature, 352: 239-241 (1991) used ratneural-specific enolase promoter inhibitor domain to prepare transgenicmice. See also, Wirak et al., Science, 253: 323-325 (1991). Still othermodels have been produced by Intracranial administration of the β/A4peptide directly to animals (Tate et al., Bull. Clin. Neurosci., 56:131-139 (1991).

It is noted that none of the in vivo animal models may turn out to beextremely good models for AD neuropathology. Instead, they may moreclosely model the amyloid deposition of normal aging. This isparticularly true of the aged-mammal models. All of these models show apreponderance of diffuse plaques as discussed above for the aged dogmodel. While there is some cerebrovascular amyloid, there are fewneuritic plaques, except in the Games et al., transgenic mouse model.The other transgenic mouse models often show only diffuse plaques.Therefore, while these models may be useful for studying distribution ofthe probes in the brain, there is a fairly low probability that thesemodels would show the same quantitative differences that would beexpected to be seen in AD brain based on the in vitro studies ofChrysamine G binding to AD brain described above.

Evaluating the Ability of Chrysamine G Derivatives to Cross the HumanBlood-Brain Barrier

A dose of approximately 10 mCi of an appropriately radiolabeledderivative of Chrysamine G with a specific activity of approximately 500Ci/mole or higher is injected intravenously into normal subjects andpatients suspected of having AD and monitored by SPECT or PET imaging toanalyze the detectability of the derivative in brain relative to otherorgans and to define the time course of detectability in the brain. Adose which can be reliably detected is defined as a “imaging effectivedose.”

Evaluation of Chrysamine G and Chrysamine G Derivatives to DistinguishAD from Age-Matched Controls in Humans

An imaging-effective dose of an appropriately, radioactively labeledderivative of Chrysamine G is injected into a subject suspected ofhaving brain amyloid deposition due to pathological conditions such asAD. After a period of 15 minutes to 24 hours, the radioactive signalfrom brain is detected by SPECT or PET. Radioactivity is simultaneouslydetected in all brain areas included in the field of view of thedetector. This field of view will be set up so as to include largeportions of the cerebellum, superior temporal cortex, superior/middlefrontal cortex, and intervening brain regions. An MRI scan will beperformed prior to the study so that corrections can be made for brainatrophy in the areas of interest by methods discussed in Example 3. TheS/V_(A), S/V_(CB), and Ratio_(A) variables discussed in Example 3 willbe calculated and compared to analogous normative ratios obtainedpreviously from age-matched normal control subjects.

EXAMPLE 6 Histologic Localization of Chrysamine G Binding toCerebrovascular Amyloid

The top frame of FIG. 11 demonstrates two neuritic plaques stained byChrysamine G. The staining method was that of Stokes and Trickey, J.Clin. Pathol. 26: 241-242 (1973) with Chrysamine G substituted for Congored. Except for somewhat lower intensity, these deposits are identicalto those stained with Congo red (not shown). The bottom two frames ofthe photomicrograph in FIG. 11 show adjacent sections from the temporallobe of an AD patient with amyloid angiopathy. The section in the lowerleft was stained with the Congo red using the method of Puchtler.Puchtler et al., J. Histochem. Cytochem. 10: 35 (1962). The section inthe lower right of FIG. 11 was stained by substituting Chrysamine G forCongo red in the Puchtler method. Both sections readily demonstrate thesame amyloid-laden vessel. A small amount of backgroundauto-fluorescence from erythrocytes and lipofuscin also is visible. Bothphotomicrographs were obtained with a laser confocal microscope usingfluorescein isothiocyanate (FITC) filters. The bar represents 20microns.

EXAMPLE 7 Assesing the Toxicity of Chrysamine G and Chrysamine GDerivatives

At doses of 10 and 100 mg/kg non-radioactive Chrysamine G administeredintraperitoneally, no notable behavioral effects or toxicity wereobserved in mice for periods up to 72 hrs. The doses of [¹⁴C]ChrysamineG administered were on the order of 1 mg/kg.

Chrysamine G appeared to show little acute toxicity based on attempts toestablish an LD₅₀. Even when the maximum volume that can be injectedinto a mouse without harming it just from fluid volume effects (approx.0.025 ml/g) of a saturated solution of Chrysamine G was injected intomice (100 mg/kg), there were no behavioral changes noted for at least 72hrs, the longest period tested. Doses required for detection ofradiolabeled derivatives by SPECT or PET would be orders of magnitudebelow this dose.

Congo red has been safely injected into humans in quantities muchgreater than would be used for the radioactive Chrysamine G derivatives.The LD₅₀ for Congo red has been shown to be 190 mg/kg mouse (Tubis etal., J. Amer. Pharm. Assoc. 49: 422 (1960)), which is similar tothe >100 mg/kg LD₅₀ shown for Chrysamine G. Thus, these two chemicallysimilar compounds cause similar low toxicities in mice.

Other Chrysamine G derivatives can similarly be tested for toxicity inmice and other higher mammals by injecting a wide range ofconcentrations and studying the animals for various signs of toxicity bymethods well known in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

EXAMPLE 8 Assesing the Ability of Chrysamine G to Protect Against Aβ(25-35)-Induced Toxicity Protection from Aβ (25-35)-Induced Toxicity inPC-12 cells

Rat pheochromocytoma cells (PC-12) were grown in RPMI 1640 media with10% fetal bovine serum. Approximately 5,000 exponentially growing cellswere plated in 96-well plates in a volume of 100 μl of media and allowedto incubate at 37° C. overnight. The Aβ (25-35), which had beenpre-aggregated at 37° C. for 7 days, was pre-incubated with Chrysamine G(CG) or related compounds in aqueous solution prior to addition of 20 μlto achieve the final concentrations given (0.01 to 10 μM Aβ (25-35) and0.03 to 20 μM CG). The cells were incubated for 24 hrs prior to theaddition of 13.3 μl of 5 mg/ml MTT(3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) in sterilephosphate buffered saline. After 4.5 hrs at 37° C., 100 μl of extractionbuffer (20% w/v SDS in 50% DMF/water; pH adjusted to 4.7 with 2.5% of80% acetic acid and 2.5% 1N HCl) was added and the plates were incubatedovernight. Hansen et al., J. Immunol. Methods 119: 203 (1989). Colordevelopment was then measured at 560 nm. Maximum viability was definedas the absorbance of control wells to which only the 20 μl of distilled,deionized H2O was added. Maximum toxicity was defined by wells in whichthe cells were lysed by addition of 0.1% (final concentration) of TritonX-100.

Incubation of PC12 cells with Aβ (25-35) results in aconcentration-dependent decrease in the ability of these cells to reduceMTT (FIG. 12). FIG. 12 shows the effect of increasing concentrations ofAβ (25-35) in the presence and absence of Chrysamine G on the cellularredox activity of PC12 cells as measured by MTT reduction. The reductionproduct of MTT absorbs at 560 nm which is plotted on the vertical axis.The effect of Aβ (25-35) alone is shown in the filled bars and shows adose dependent decrease in MTT reduction. Significant differences fromcontrol (no Aβ, no Chrysamine G) are shown in white numbers inside thefilled bars. The protective effect of 20 μM Chrysamine G is shown in theopen bars. Significant differences between MTT reduction in the presenceand absence of Chrysamine G are shown in black numbers inside the openbars.

FIG. 13 demonstrates the protective effect of increasing concentrationsof Chrysamine G against the Aβ (25-35)-induced reduction of cellularredox activity of PC12 cells. The effect of Chrysamine G in the absenceof Aβ (25-35) is shown in the filled bars. There was no significantdifference between control (no Aβ, no Chrysamine G) and any of theconcentrations of Chrysamine G in the absence of Aβ (25-35). MTTreduction in the presence of 1 μM Aβ (25-35) and increasingconcentrations of Chrysamine G is shown in the open bars. Significantdifferences in MTT reduction between the presence and absence of Aβ(25-35) at each concentration of Chrysamine G are shown in white numbersinside the filled bars. Significant differences in MTT reduction betweenthe Aβ (25-35) control (no Chrysamine G) and Aβ (25-35) plus increasingconcentrations of Chrysamine G are shown in black numbers inside theopen bars.

As has previously been reported, Congo red protects against theAβ-induced toxicity at concentrations over 2 μM, achieving completeprotection by 20 μM. Burgevin et al. NeuroReport 5: 2429 (1994); Lorenzoand Yankner, Proc. Natl. Acad. Sci. 91: 12243 (1994); Pollack et al.,Neuroscience Letters 184: 113 (1995); Pollack et al.Neuroscience Letters197: 211 (1995). Chrysamine G shows a protective effect which isdependent on both the concentration of Aβ (25-35) (FIG. 12) as well asthe concentration of Chrysamine G (FIG. 13). The protective effect ofChrysamine G is evident at 0.2 μM, a concentration very close to the Kiof Chrysamine G for binding to synthetic Aβ, 0.37 μM (FIG. 1).Chrysamine G appears to be more potent than Congo red, showing effectsin the range of 0.1 to 1.0 μM. This is consistent with the Ki values forbinding to synthetic Aβ of 0.37 μM for Chrysamine G and 2.8 μM for Congored (FIG. 1).

In another experiment (FIG. 14), the effect of Chrysamine G and thephenol derivative (see FIG. 1), which does not bind Aβ, was examined incells incubated with 1 μM Aβ (25-35). Chrysamine G showed protectiveeffects at 0.1 and 1 μM, but the phenol derivative showed no protectiveeffects, and perhaps enhanced the toxicity of Aβ.

These results suggest that the lipophilic derivative of Congo red,Chrysamine G, prevents Aβ-induced cytotoxicity in cell culture atconcentrations very similar to those at which it binds Aβ. Thisprotection shows structural specificity since the phenol derivativewhich does not bind to synthetic Aβ also does not prevent Aβ -inducedcytotoxicity. Since Chrysamine G partitions into the brain well, theseresults provide evidence that Chrysamine G and Aβ-binding derivatives ofChrysamine G have therapeutic potential in the treatment of AD.

The mechanism of the protective effect of Chrysamine G is unknown atpresent. Two broad possibilities exist. First, Chrysamine G couldinterfere with the aggregation of Aβ. Second, Chrysamine G couldinterfere with the effects (direct or indirect) of Aβ on the targetcells. Congo red does inhibit aggregation of Aβ as well as protectagainst the toxic effects of aggregated Aβ. Lorenzo and Yankner, Proc.Natl. Acad. Sci. 91: 12243 (1994). Interference with aggregation isunlikely in the above experiment since the Aβ was pre-aggregated priorto incubation with Chrysamine G. Thus, inhibition of aggregation couldprove to be an important therapeutic effect of Chrysamine G but is not alikely explanation for the protective effects of Chrysamine G againstpre-aggregated Aβ. The model of Chrysamine G binding to Aβ described inFIG. 7, displays how Chrysamine G could “coat” the surface of Aβ. Thismay change how the fibrillar deposits are recognized by cell-surfacereceptors or other macromolecules such as complement proteins andinterfere with the toxic effects of Aβ which may be mediated by thesemacromolecules. It is likely that Chrysamine G and Congo red exertmultiple effects, both before and after the aggregation of Aβ. This isadvantageous from a therapeutic point of view since patients are likelyto present at a time when there are pre-existing Aβ aggregates as wellas ongoing amyloid deposition.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention. It is intended that the specification be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

What is claimed is:
 1. An amyloid binding compound of Formula I or awater soluble, non-toxic salt thereof:

wherein:

is selected from the group consisting of N═N—Q, CR′═N—Q, N═CR′—Q,CR′₂—NR′—Q, NR′—CR′₂—Q, (CO)—NR′—Q, NR′—(CO)—Q and NR′—NR′—Q (where R′independently represents H or a lower alkyl group); X is C(R″)₂,(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),(where R_(ph) represents an unsubstituted or substituted phenyl groupwith the phenyl substituents being chosen from any of the non-phenylsubstituents defined from R″), a tetrazole or oxadiazole of the form:

wherein R′ is H or a lower alkyl group); or X is CR′═CR′, N═N, C═O, O,NR′, (where R′ represents H or a lower alkyl group), S, or SO₂; each R₁and R₂ independently is selected from the group consisting of H, F, Cl,Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂), tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

 (wherein R₈ and R₉ are lower alkyl groups) or

and at least one of R₂ is not H, OCH₃, CH₃ or halogen; each Q isindependently selected from one of the following structures, each ofwhich contains a carboxylic acid or an acid like functionality selectedfrom the group consisting of hydroxy, sulfhydryl, tetrazole, oxadiazole,isoindol-1,3 (2H)-dione, benzisoxazole, 2,3-benzodiazine-1,4(2H,3H)-dione, 2,3-benzoxazine-1,4 (3H)-dione, (2H) 1,3-benzoxazine-2,4(3H)-dione, (3H) 2-benzazine-1,3 (2H)-dione, and NO₂; IA, IB, IC, ID,IE, IF, and IG, wherein IA has the following structure:

 wherein: each of R₃, R₄, R₅, R₆, or R₇ is independently defined thesame as R₁ above and wherein at least one of R₃, R₄, R₅, R₆, or R₇ is ahydroxy, tetrazole, oxadiazole, NO₂, or carboxy in both Q's; wherein R₁or R₂ is not H or COOR′ when R₆ is COOR′; and R₁ or R₂ is not H when R₅or R₇ is OH; and R₂ is not COOH when R₃=OH, R₄=COOR′or R₅=NH₂ IB has thefollowing structure:

 wherein: each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently isdefined the same as R₁ above above and, wherein at least one of R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ is a hydroxy, tetrazole, oxadiazole,NO₂, or carboxy in both Q's and wherein at least one of R₁ or R₂ is ahalogen when the compound of Formula I is a 4,4′-diazophenyl compound;provided that if the Formula IB moiety is:

then at least one of R₁ or R₂ is Br or I or at least one or R₁₁-R₁₅ isnot H, bromo or methoxy; IC has the following structure:

 wherein: each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁ independently is definedthe same as R₁ above; ID has the following structure:

wherein each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁above and

represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein: each of R₂₅, R₂₆, or R₂₇ independently is defined the same asR₁ above and

represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein: exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 of the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁, orR₃₂ independently is defined the same as R₁ above, and wherein at leastone of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂, or carboxy in both Q's; IG has the following structure:

 wherein: exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is the

 of the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈ or R₃₉ independently is defined the same as R₁ above andwherein at least one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's;or wherein:

is NR′—N═Q (wherein R′ represents H or a lower alkyl group) and each

 is independently selected from one of the following structures: IIA orIIB, wherein: IIA has the following structure

 wherein each of R₄₀-R₄₇ independently is H, F, Cl, Br, I, a lower alkylgroup, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SH, COOR′, a tri-alkyl tin, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R₁), a tri-alkyl tin, atetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group); IIB has the followingstructure:

 wherein: each of R₄₈ and R₄₉ independently is a lower alkyl group,(CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, CH₂—CH₂—CH₂F, CN,(C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, (C═O)—CH₂)_(n)—(C₆H₅) where n=1, 2,or 3, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) representsan unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R₁), a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group, wherein at least one of R₄₈ orR₄₉ is (C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, CN, or a tetrazole oroxadiazole of the form:

(wherein R′ is H or lower alkyl group) in both Q's.
 2. The compound ofclaim 1, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉ isselected from the group consisting of R_(ph), CR′═CR′—R_(ph),CR′₂—CR′₂—R_(ph).
 3. The compound of claim 1, wherein at least one ofthe substituents R₁-R₇ and R₁₀-R₄₉ is selected from the group consistingof ¹³¹I, 123I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—¹⁸F, O—CH₂—CH₂—¹⁸F,CH₂—CH₂—CH₂—¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F, ¹⁹F, ¹²⁵I and a ¹¹C or ¹³C labeledmoiety selected from the group consisting of a lower alkyl group,(CH₂)_(n)OR′, CF₃, CH₂—CH₂—F, O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂,COOR′, R_(ph), CR′═CR′—R_(ph), and CR′₂—CR′₂R_(ph=), wherein n, R′ andR_(ph) are as defined in claim
 1. 4. The compound of claim 1, whereinsaid compound binds to Aβ with a dissociation constant (K_(D)) between0.0001 and 10.0 μM when measured by binding to synthetic Aβ peptide orAlzheimer's Disease brain tissue.
 5. A method for synthesizing acompound of claim 1, wherein at least one of the substituents R₁-R₇ andR₁₀-R₄₉ is selected from the group consisting of ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F, and ¹⁹F, comprising the step of labeling a compound of claim1, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉ is atri-alkyl tin or a triazene, by reaction of a compound of claim 1 with aradiohalogenating compound having a radiolabel selected from the groupconsisting of ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, or ¹⁹F.
 6. Apharmaceutical composition for in vivo imaging of amyloid deposits,comprising (a) a compound of claim 3 or a salt thereof, and (b) apharmaceutically acceptable carrier.
 7. An in vivo method for detectingamyloid deposits in a subject, comprising the steps of: (a)administering a detectable quantity of radiolabeled Chrysamine G or aChrysamine G derivative wherein R₁-R₇ or R₁₀-R₄₉ are radiolabeled andare selected from the group consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, 18F,CH₂—CH₂ 13 ¹⁸F, O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂ 13 ¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F,¹⁹F, ¹²⁵I and a ¹¹C or ¹³C labeled moeity selected from the groupconsisting of a lower alkyl group, (CH₂)_(n)OR′, CF₃, CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CH₂—CH₂—CH₂—F, O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂, COOR′, R_(ph), CR′═CR′—R_(ph),and CR′₂—CR′₂R_(ph=), wherein n, R′ and R_(ph) are as defined below, and(b) detecting the binding of said compound or a salt thereof to amyloiddeposit in said subject; wherein said Chrysamine G derivative is anamyloid binding compound of Formula I or a water soluble toxic saltthereof:

wherein:

is selected from the group consisting of N═N—Q, CR′═N—Q, N═CR′—Q,CR′₂—NR′—Q, NR′—CR′₂—Q, (CO)—NR′—Q, NR′—(CO)—Q and NR′—NR′—Q (where R′independently represents H or a lower alkyl group); X is C(R″)₂,(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),(where R_(ph) represents an unsubstituted or substituted phenyl groupwith the phenyl substituents being chosen from any of the non-phenylsubstituents defined from R″), a tetrazole or oxadiazole of the form:

wherein R′ is H or a lower alkyl group); or X is CR′═CR′, N═N, C═O, O,NR′, (where R′ represents H or a lower alkyl group), S, or SO₂; each R₁and R₂ independently is selected from the group consisting of H, F, Cl,Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂), tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

 (wherein R₈ and R₉ are lower alkyl groups) or

and at least one of R₂ is not H, OCH₃, CH₃ or halogen; each Q isindependently selected from one of the following structures, each ofwhich contains a carboxylic acid or an acid like functionality selectedfrom the group consisting of hydroxy, sulfhydryl, tetrazole, oxadiazole,isoindol-1,3 (2H)-dione, benzisoxazole, 2,3-benzodiazine-1,4(2H,3H)-dione, 2,3-benzoxazine-1,4 (3H)-dione, (2H) 1,3-benzoxazine-2,4(3H)-dione, (3H) 2-benzazine-1,3 (2H)-dione, and NO₂: IA, IB, IC, ID,IE, IF, and IG, wherein IA has the following structure:

 wherein: each of R₃, R₄, R₅, R₆, or R₇ is independently defined thesame as R₁ above and wherein at least one of R₃, R₄, R₅, R₆, or R₇ is ahydroxy, tetrazole, oxadiazole, NO₂, or carboxy in both Q's; IB has thefollowing structure:

 wherein: each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently isdefined the same as R₁ above and, wherein at least one of R₁₀, R₁₁, R₁₂,R₁₃, R₁₄, R₁₅, or R₁₆ is a hydroxy, tetrazole, oxadiazole, NO₂, orcarboxy in both Q's; and wherein at least one of R₁ or R₂ is a halogenwhen the compound of Formula I is a 4,4′-diazobiphenyl compound; IC hasthe following structure:

 wherein: each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁ independently is definedthe same as R₁ above; ID has the following structure:

wherein each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁above and

represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein: each of R₂₅, R₂₆, or R₂₇ independently is defined the same asR₁ above and

represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein: exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 of the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁, orR₃₂ independently is defined the same as R₁ above, and wherein at leastone of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂, or carboxy in both Q's; IG has the following structure:

 wherein: exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is the

 of the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈ or R₃₉ independently is defined the same as R₁ above andwherein at least one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's;or wherein:

is NR′—N═Q (wherein R′ represents H or a lower alkyl group) and each

 is independently selected from one of the following structures: IIA orIIB, wherein: IIA has the following structure

 wherein each of R₄₀-R₄₇ independently is H, F, Cl, Br, I, a lower alkylgroup, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SH, COOR′, a tri-alkyl tin, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R₁), a tri-alkyl tin, atetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group); IIB has the followingstructure:

 wherein: each of R₄₈ and R₄₉ independently is a lower alkyl group,(CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, CH₂—CH₂—CH₂F, CN,(C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, (C═O)—CH₂)_(n)—(C₆H₅) where n=1, 2,or 3, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) representsan unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R₁), a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group, wherein at least one of R₄₈ orR₄₉ is (C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, CN, or a tetrazole oroxadiazole of the form:

(wherein R′ is H or lower alkyl group) in both Q's.
 8. The method ofclaim 7, wherein said amyloid deposit is located in the brain of asubject.
 9. The method of claim 7, wherein said subject is suspected ofhaving a disease or syndrome selected from the group consisting ofAlzheimer's Disease, familial Alzheimer's Disease, Down's Syndrome andhomozygotes for the apolipoprotein E4 allele.
 10. The method of claim 7,wherein said detecting is selected from the group consisting of gammaimaging, magnetic resonance imaging and magnetic resonance spectroscopy.11. The method of claim 10, wherein said gamma imaging is either PET orSPECT.
 12. The method of claim 7, wherein said pharmaceuticalcomposition is administered by intravenous injection.
 13. The method ofclaim 8, wherein the ratio of (i) binding of said compound to a brainarea other than the cerebellum to (ii) binding of said compound to thecerebellum, in said subject, is compared to said ratio in normalsubjects.
 14. The compound of claim 1, wherein the acid likefunctionality is provided by a functional group which contains anionizable proton with a pK_(a) of less than
 10. 15. A method ofinhibiting cell degeneration and toxicity associated with fibrilformation in an amyloidosis-associated condition, said method comprisingthe step of administering to a subject having or suspected of havingsuch condition a pharmaceutically effective amount of Chrysamine G or aderivative thereof, wherein said derivative of Chrysamine G is anamyloid binding compound of Formula I or a water soluble, non-toxic saltthereof

wherein:

is selected from the group consisting of N═N—Q, CR′═N—Q, N═CR′—Q,CR′₂—NR′—Q, NR′—CR′₂—Q, (CO)—NR′—Q, NR′—(CO)—Q and NR′—NR′—Q (where R′independently represents H or a lower alkyl group); X is C(R″)₂,(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),(where R_(ph) represents an unsubstituted or substituted phenyl groupwith the phenyl substituents being chosen from any of the non-phenylsubstituents defined from R″), a tetrazole or oxadiazole of the form:

wherein R′ is H or a lower alkyl group); or X is CR′═CR′, N═N, C═O, O,NR′, (where R′ represents H or a lower alkyl group), S, or SO₂; each R₁and R₂ independently is selected from the group consisting of H, F, Cl,Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂), a tetrazole or a oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

 (wherein R₈ and R₉ are lower alkyl groups) or

and at least one of R₂ is not H, OCH₃, CH₃, or halogen; each Q isindependently selected from one of the following structures, each ofwhich contains a carboxylic acid or an acid like functionality selectedfrom the group consisting of hydroxy, sulfhydryl, tetrazole, oxadiazole,isoindol-1,3 (2H)-dione, 3-hydroxy-1,2-benzisoxazole,2,3-benzodiazine-1,4 (2H,3H)-dione, 2,3-benzoxiaine-1,4 (3H)-dione, (2H)1,3-benzoxazine-2,4 (3H)-dione, (3H) 2-benzazine-1,3 (2H)-dione, andNO₂: IA, IB, IC, ID, IE, IF, and IG, wherein IA has the followingstructure:

 wherein: each of R₃, R₄, R₅, R₆, or R₇ independently is defined thesame as R₁ above and, wherein at least one of R₃, R₄, R₅, R₆, or R₇ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's,and wherein at least one of R₁ or R₂ is a halogen when the compound ofFormula I is a 4,4′-diazobiphenyl compound; IB has the followingstructure:

 wherein: each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently isdefined the same as R₁ above above and, wherein at least one of R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂, or carboxy in both Q's, and wherein at least one of R₁,or R₂ is a halogen when the compound of Formula I is a4,4′-diazobiphenyl compound; IC has the following structure:

 wherein: each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁, independently is definedthe same as R₁ above; ID has the following structure:

wherein each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁above and

represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein: each of R₂₅, R₂₆, or R₂₇ independently is defined the same asR₁ above and

represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein: exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 of the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁, orR₃₂ independently is defined the same as R₁ above, and wherein at leastone of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂, or carboxy in both Q's; IG has the following structure:

 wherein: exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is the

 of the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈ or R₃₉ independently is defined the same as R₁ above andwherein at least one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's;or wherein:

is NR′—N═Q (wherein R′ represents H or a lower alkyl group) and each

 is independently selected from one of the following structures: IIA orIIB, wherein: IIA has the following structure

 wherein: each of R₄₀-R₄₇ independently is H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F,O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂,(C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R₁), atri-alkyl tin, a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group); IIB has the followingstructure:

 wherein: each of R₄₈ and R₄₉ independently is a lower alkyl group,(CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, CH₂—CH₂—CH₂F, CN,(C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, (C═O)—CH₂)_(n)—(C₆H₅) where n=1, 2,or 3, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) representsan unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R₁), a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), wherein at least one of R₄₈ orR₄₉ is (C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, CN, or a tetrazole oroxadiazole of the form:

(wherein R′ is H or lower alkyl group) in both Q's.
 16. The method ofclaim 15, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉ isR_(Ph), CR′═CR′—R_(Ph), CR′₂—CR′₂—R_(Ph).
 17. The method of claim 15wherein each R₂ is CH₃ when

is N═N—Q.
 18. The method of claim 15, wherein saidamyloidosis-associated condition is selected from the group consistingof Alzheimer's Disease, Down's Syndrome, Type 2 diabetes mellitus,hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A(reactive), secondary amyloidosis, familial mediterranean fever,familial amyloid nephropathy with urticaria and deafness (Muckle-wellsSyndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic,myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele, and the condition associated withhomozygosity for the apolipoprotein E4 allele or Huntington's disease.19. The method of claim 15, wherein the acid-like functionality isprovided by a functional group which contains an ionizable proton with apK_(a) of less than
 10. 20. A pharmaceutical composition for theprevention of cell degeneration and toxicity associated with fibrilformation in amyloidosis-associated conditions comprising the compoundof claim 1 or a salt thereof and a pharmaceutically acceptable carrier.21. A method of detecting amyloid deposits in biopsy or post-mortemhuman or animal tissue comprising the steps of: (a) incubatingformalin-fixed tissue with a solution of a Chrysamine G or a ChrysamineG derivative or a salt thereof to form a labelled deposit and then, (b)detecting the labelled deposits, wherein said Chrysamine G derivative isas defined in claim
 7. 22. The method of claim 21 wherein said solutionis composed of 25-100% ethanol (the remainder being water) saturatedwith the compound of claim 1 or a salt thereof.
 23. The method of claim21 wherein said detecting is effected by microscopic techniques selectedfrom the group consisting of bright-field, fluorescence, laser-confocal,and cross-polarization microscopy.
 24. The method of claim 7, wherein atleast one of the substituents R₁-R₇ and R₁₀-R₄₉ is selected from thegroup consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—¹⁸F,O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂—¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F, ¹⁹F, ¹²⁵I, and a ¹¹Cor ¹³C labeled moiety selected from the group consisting of a loweralkyl group, (CH₂)_(n)OR′, CF₃, CH₂—CH₂—F, O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂,COOR′, R_(ph), CR′═CR′—R_(ph), and CR′₂—CR′₂R_(ph=), wherein n, R′ andR_(ph) are defined in claim
 7. 25. A method of quantifying the amount ofamyloid in biopsy or post-mortem tissue comprising the steps of: a)incubating radiolabeled Chrysamine G or a derivative of Chrysamine Gwith a homogenate of biopsy or post-mortem tissue, wherein R₁-R₇ orR₁₀-R₄₉ are radiolabeled and are selected from the group consisting of¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—¹⁸F, O—CH₂—CH₂—¹⁸F,CH₂—CH₂—CH₂—¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F, ¹⁹F, ¹²⁵I and a ¹¹C or ¹³C labeledmoeity selected from the group consisting of a lower alkyl group,(CH₂)_(n)OR′, CF₃, CH₂—CH₂—F, O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂,COOR′, R_(ph), CR′═CR′—R_(ph), and CR′₂—CR′₂R_(ph=), wherein n, R′ andR_(ph) are as defined below, b) separating the tissue-bound from thetissue-unbound radiolabeled Chrysamine G or Chrysamine G derivative, c)quantifying the tissue-bound radiolabeled Chrysamine G or Chrysamine Gderivative, and d) converting the units of tissue-bound radiolabeledChrysamine G or Chrysamine G derivative to units of micrograms ofamyloid per 100 mg of tissue by comparison with a standard wherein saidChrysamine G derivative is an amyloid binding compounds of Formula I ora water soluble toxic salt thereof:

wherein:

is selected from the group consisting of N═N—Q, CR′═N—Q, N═CR′—Q,CR′₂—NR′—Q, NR′—CR′₂—Q, (CO)—NR′—Q, NR′—(CO)—Q and NR′—NR′—Q (where R′independently represents H or a lower alkyl group); X is C(R″)₂,(wherein each R″ independently is H, F, Cl, Br, I, a lower alkyl group,(CH₂)_(n)OR′ where n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph),(where R_(ph) represents an unsubstituted or substituted phenyl groupwith the phenyl substituents being chosen from any of the non-phenylsubstituents defined from R″), a tetrazole or oxadiazole of the form:

wherein R′ is H or a lower alkyl group); or X is CR′═CR′, N═N, C═O, O,NR′, (where R′ represents H or a lower alkyl group), S, or SO₂; each R₁and R₂ independently is selected from the group consisting of H, F, Cl,Br, I, a lower alkyl group, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃,CH₂—CH₂F, O—CH₂—CH₂F, CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, a tri-alkyl tin,R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined from R₁ andR₂), tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group), or a triazene of the form:

 (wherein R₈ and R₉ are lower alkyl groups) or

and at least one of R₂ is not H, OCH₃, CH₃ or halogen; each Q isindependently selected from one of the following structures, each ofwhich contains a carboxylic acid or an acid like functionality selectedfrom the group consisting of hydroxy, sulfhydryl, tetrazole, oxadiazole,isoindol-1,3 (2H)-dione, benzisoxazole, 2,3-benzodiazine-1,4(2H,3H)-dione, 2,3-benzoxazine-1,4 (3H)-dione, (2H) 1,3-benzoxazine-2,4(3H)-dione, (3H) 2-benzazine-1,3 (2H)-dione, and NO₂: IA, IB, IC, ID,IE, IF, and IG, wherein IA has the following structure:

 wherein: each of R₃, R₄, R₅, R₆, or R₇ is independently defined thesame as R₁ above and wherein at least one of R₃, R₄, R₅, R₆, or R₇ is ahydroxy, tetrazole, oxadiazole, NO₂, or carboxy in both Q's; IB has thefollowing structure:

 wherein: each of R₁₀, R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ independently isdefined the same as R₁ above above and, wherein at least one of R₁₀,R₁₁, R₁₂, R₁₃, R₁₄, R₁₅, or R₁₆ is a hydroxy, tetrazole, oxadiazole,NO₂, or carboxy in both Q's and wherein at least one of R₁ or R₂ is ahalogen when the compound of Formula I is a 4,4′-diazophenyl compound;IC has the following structure:

 wherein: each of R₁₇, R₁₈, R₁₉, R₂₀, or R₂₁ independently is definedthe same as R₁ above; ID has the following structure:

wherein each of R₂₂, R₂₃, or R₂₄ independently is defined the same as R₁above and

represents a heterocyclic ring of one of the six following formulas:

IE has the following structure:

 wherein: each of R₂₅, R₂₆, or R₂₇ independently is defined the same asR₁ above and

represents a heterocyclic ring of one of the six following formulas:

IF has the following structure:

 wherein: exactly one of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is the

 of the

 link defined for Formula I above and each other R₂₈, R₂₉, R₃₀, R₃₁, orR₃₂ independently is defined the same as R₁ above, and wherein at leastone of R₂₈, R₂₉, R₃₀, R₃₁, or R₃₂ is a hydroxy, sulfhydryl, tetrazole,oxadiazole, NO₂, or carboxy in both Q's; IG has the following structure:

 wherein: exactly one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is the

 of the

 link defined for Formula I above and each other R₃₃, R₃₄, R₃₅, R₃₆,R₃₇, R₃₈ or R₃₉ independently is defined the same as R₁ above andwherein at least one of R₃₃, R₃₄, R₃₅, R₃₆, R₃₇, R₃₈ or R₃₉ is ahydroxy, sulfhydryl, tetrazole, oxadiazole, NO₂, or carboxy in both Q's;or wherein:

is NR′—N═Q (wherein R′ represents H or a lower alkyl group) and each

 is independently selected from one of the following structures: IIA orIIB, wherein: IIA has the following structure

 wherein each of R₄₀-R₄₇ independently is H, F, Cl, Br, I, a lower alkylgroup, (CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, O—CH₂—CH₂F,CH₂—CH₂—CH₂F, O—CH₂—CH₂—CH₂F, CN, (C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂,O(CO)R′, OR′, SH, COOR′, a tri-aLkyl tin, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph), (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R₁), a tri-alkyl tin, atetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group); IIB has the followingstructure:

 wherein: each of R₄₈ and R₄₉ independently is a lower alkyl group,(CH₂)_(n)OR′, wherein n=1, 2, or 3, CF₃, CH₂—CH₂F, CH₂—CH₂—CH₂F, CN,(C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, (C═O)—CH₂)_(n)—(C₆H₅) where n=1, 2,or 3, R_(ph), CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph), (where R_(ph) representsan unsubstituted or substituted phenyl group with the phenylsubstituents being chosen from any of the non-phenyl substituentsdefined for R₁), a tetrazole or oxadiazole of the form:

(wherein R′ is H or a lower alkyl group, wherein at least one of R₄₈ orR₄₉ is (C═O)—R′, NO₂, (C═O)N(R′)₂, COOR′, CN, or a tetrazole oroxadiazole of the form:

(wherein R′ is H or lower alkyl group) in both Q's.
 26. The method ofclaim 25, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉ isselected from the group consisting of R_(ph), CR′═CR′—R_(Ph),CR′₂—CR′₂—R_(Ph) (where R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R₁).
 27. The method ofclaim 25, wherein at least one of the substituents R₁-R₇ and R₁₀-R₄₉ islabeled with a radiolabel selected from the group consisting of ¹²⁵I, ³Hand a labeled moiety selected from the group consisting of a lower alkylgroup, (CH₂)_(n)OR′, CF₃, CH₂—CH₂—F, O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂,COOR′, R_(ph), CR′═CR′—R_(ph), and CR′₂—CR′₂—R_(ph), wherein at leastone carbon of the labeled moiety is ¹⁴C.
 28. A method for synthesizing abiphenyl compound of claim 1, wherein R₂ is ³H, comprising performing areaction of 3,3′-[³H]benzidine with a selected coupling component toyield the compound of claim
 1. 29. A method of distinguishing anAlzheimer's disease brain from a normal brain comprising: a) separatelyincubating homogenates of weighed tissue from (i) the cerebellum and(ii) another area of the same brain other than the cerebellum, from asubject suspected of having Alzheimer's disease, with a radiolabeledChrysamine G or a radiolabeled Chrysamine G derivative so that amyloidin each said tissue binds with said radiolabeled Chrysamine G or aradiolabeled Chrysamine G derivative; b) quantifying the amount ofamyloid bound to said radiolabeled Chrysamine G or a radiolabeledChrysamine G derivative by; b1) separating the tissue-bound from thetissue-unbound radiolabeled Chrysamine G or a radiolabeled Chrysamine Gderivative, b2) quantifying the tissue-bound radiolabeled Chrysamine Gor a radiolabeled Chrysamine G derivative, and b3) converting the unitsof tissue-bound radiolabeled Chrysamine G or a radiolabeled Chrysamine Gderivative to units of micrograms of amyloid per 100 mg of tissue bycomparison with a standard, c) calculating the ratio of the amount ofamyloid in the area of the brain other than the cerebellum to the amountof amyloid in the cerebellum; d) comparing the ratio of the amount ofamyloid in tissue from the subject suspected of having Alzheimer'sdisease with ratios for the amount of amyloid in said tissue from normalsubjects; and e) determining the presence of Alzheimer's disease if saidratio from the brain of a subject suspected of having Alzheimer'sdisease is above 90% of the ratios obtained from the brains of normalsubjects, wherein said radiolabeled Chrysamine G derivative is anamyloid binding compound of Formula I or a water soluble, non-toxic saltthereof, wherein R₁-R₇ or R₁₀-R₄₉ are radiolabeled and are selected fromthe group consisting of ¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—¹⁸F,O—CH₂—CH₂—¹⁸F, CH₂—CH₂—CH₂—¹⁸F, O—CH₂—CH₂—CH₂—¹⁸F, ¹⁹F, ¹²⁵I, and a ¹¹C,or ¹³C labeled moiety selected from the group consisting of a loweralkyl group, (CH₂)_(n)OR′, CF₃, CH₂—CH₂—F, O—CH₂—CH₂—F, CH₂—CH₂—CH₂—F,O—CH₂—CH₂—CH₂—F, CN, (C═O)—R′, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, N(R′)₂,COOR′, R_(ph), CR′═CR′—R_(ph), and CR′₂—CR′₂R_(ph=), wherein n, R′ andR_(ph) are as defined in claim
 7. 30. A method according to claim 28,wherein the reaction is selected from the group consisting of azocoupling, amide coupling, or Schiff base formation, and wherein theselected coupling component is selected from the group consisting of asalicylic acid derivative, or a naphthoic acid derivative.
 31. Themethod of claim 25, wherein the acid-like functionality is provided by afunctional group which contains an ionizable proton with a pK_(a) ofless than